CN115804847A - PH/hydrogen peroxide/MMP 9 time-ordered response microsphere, exosome-carrying biological carrier and application - Google Patents

Abstract

The invention discloses a pH/hydrogen peroxide/MMP 9 time-sequence response microsphere and an exosome-carrying biological carrierBody and application, relate to medicine carrier technical field. The exosome derived from regulatory T cells based on SPARC high expression has the protection effect on the heart function, and the pH and H in the infarct area ₂ O ₂ And MMP9 level, synthesis of conjugated peptide, pH/H ₂ O ₂ MMP9 sequential response microspheres and an exosome-carrying biological carrier. First, an exosome and a hydrogel raw material are released by cleaving an acylhydrazone bond under acidic conditions. Subsequently, using H ₂ O ₂ Mixing Co ²⁺ Oxidation to Co ³⁺ Forming a gel and immobilizing the released exosomes. Finally, the gel gradually degrades upon hydrolysis of MMP9, releasing exosomes, thereby exerting a sustained and long-term protective effect on the myocardium to improve cardiac function.

Description

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

Technical Field

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

Background

In Acute Myocardial Infarction (AMI), regulatory T cells (Tregs) can suppress differentiated CD4 ⁺ T cell, CD8 ⁺ Effector activity of T cells, th17 cells, and function of natural killer and B cells, can also influence the healing process of injury by modulating monocyte/macrophage differentiation [1]. Tregs infiltrating into myocardium highly express SPARC (cysteine-rich acidic secreted protein), which can increase collagen content and maturity in scar of myocardial infarction to prevent heart rupture after myocardial infarction and improve survival rate [2 ]]. Thus, spark ^high Tregs play an important protective role in tissue repair after acute myocardial infarction. Although Tregs may infiltrate into the infarcted area after myocardial infarction, it peaked at day 7, suggesting that Sparc is promoted in advance ^high Treg peaks in the infarct zone may contribute to repair of the infarct zone and reduce infarct size, 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]。

Exosomes derived from cells have functions similar to cells, which can promote angiogenesis, reduce apoptosis, inhibit fibrosis, regulate immune response, and the like [4]. It was demonstrated that exosomes secreted by dendritic cells following stimulation with 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 resident in situ in myocardial tissue limits their effectiveness in treating acute myocardial infarction. At present, genetic engineering is widely used for improving the targeting and stability of exosomes [6]. Meanwhile, many studies have demonstrated that hydrogels are good matrices for the immobilization of exosomes [7]. However, many current studies of hydrogel encapsulated exosomes are injected in situ, which can cause secondary damage to myocardial tissue. Therefore, there is an urgent need to develop a hydrogel-exosome system compatible with the local microenvironment after acute myocardial infarction to target the infarct site. The hydrogel which can be crosslinked in situ to form gel to fix exosomes and can slowly release exosomes can provide a potential scheme for treating myocardial infarction.

Analysis of local microenvironment change after acute myocardial infarction shows that in the first stage after aseptic inflammation reaction is activated, the damaged myocardium releases injury related molecular components and binds to Toll-like receptor to start the production of chemotactic factor, such as CXCL1, CXCL2, CXCL5, etc. the ligand is CXCR2, and neutral granulocyte and Ly6C inducing CXCR2 to express high ^high Recruitment of monocytes releases large amounts of proinflammatory factors. At the same time, the continuous drop in the pH of the infarcted area (pH) is caused by the continuous death and rupture of the cells, accompanied by the continuous accumulation of lactic acid<6.8)[8]Forming an acidic microenvironment [9,10 ]]. In the second phase, inflammatory cells chemotactic to the infarct area exert a pro-inflammatory effect that promotes the production of large numbers of reactive oxygen species, including H ₂ O ₂ Superoxide and hydroxyl radical, etc., further damaging the damaged myocardium [10]. Third stage, ly6C ^low Monocytes and M2-type macrophages exert an anti-inflammatory effect in an environment enriched for IL-10, TGF-beta and VEGF, while releasing matrix metalloproteinase 9 (MMP 9) [11 ]]. In the fourth stage, fibroblasts are activated and migrate to the infarcted area, where they are transformed into myofibroblasts under the action of chemokines and growth factors, and gradually accumulateThe enzymatic reaction of MMP9 is activated, degrading the extracellular matrix, promoting proliferation of myofibroblasts, promoting scar tissue formation [12]。

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

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

The invention utilizes the inflammatory reaction process after acute myocardial infarction and passes through an acidic microenvironment (pH)<6.8 To release hydrogel material (microsphere rupture, exosome release) and subsequently utilize post-myocardial infarction H ₂ O ₂ The high-concentration environment in situ forms hydrogel to fix exosomes, and then MMP9 with continuously increased content is utilized to degrade the hydrogel and release the exosomes, so that the function of repairing cardiovascular tissues is exerted.

The invention is realized by the following steps:

the invention provides a conjugated peptide, which comprises 4A-PEG modified by polypeptide and metal cobalt ions, wherein the polypeptide comprises histidine and a peptide fragment for MMP9 enzymatic hydrolysis, and the peptide fragment for MMP9 enzymatic hydrolysis has the following SEQ ID NO:2, metal cobalt ions are combined with histidine by a coordination bond; metallic cobalt ion can be substituted by H ₂ O ₂ The oxidation causes the conjugated peptide to gel.

SEQ ID NO:2 is as follows: GGALGLPG.

Previous studies have shown that in the synthesis of histidine-modified 4A-PEG aqueous solutions by peptide chemistry, the amino group in histidine and Co ²⁺ Coordinate bonds may be formed. At H ₂ O ₂ Under the action of (C), co ²⁺ Is oxidized into Co ³⁺ While the 4A-PEG aqueous solution changes from liquid to gel-like [14 ]]. H produced in the second stage of the inflammatory reaction ₂ O ₂ And MMP9 secreted in the fourth stage accumulates continuously in the infarct zone [10-12]MMP9 can specifically recognize and hydrolyze the peptide fragment "GALLGLP" [15]. The change of local microenvironment at different stages after acute myocardial infarction is utilized to ensure that the product has the function of H ₂ O ₂ By action of MMP9, forming a gelHydrolysis characteristics under action, the inventors used polypeptides (including histidine and a peptide fragment recognized and hydrolyzed by MMP9, i.e. GGALGLPGH) to modify 4A-PEG, binding Co based on metal-ligand interaction ²⁺ Finally, 4A-PEG conjugated peptide (4 APPC) is formed. The synthetic structure of 4APPC is shown in FIG. S3A.

In addition, it was verified that the above-mentioned conjugated peptide has good fluidity, once it is H ₂ O ₂ Triggered, the material exhibits good toughness and strength, which will help it adapt to the beating characteristics of the heart and achieve long-term retention. And once the material is triggered again by MMP9, the toughness and toughness characteristics disappear and the fluidity increases, thus ensuring the release of the loaded agent. The above-mentioned conjugated peptides are based on H ₂ O ₂ Responsive to design, and Co ²⁺ The coordination-linked 4A-PEG can form hydrogel in situ in the infarct area to immobilize exosomes. The peptide segment for MMP9 enzyme hydrolysis is arranged in the conjugated peptide, so that the formed hydrogel is gradually degraded and exosomes are slowly released under the action of MMP9 enzyme.

The invention also provides a pH/H ₂ O ₂ A MMP9 time-ordered response microsphere comprising: the microsphere is formed by distearoyl phosphatidyl ethanolamine and polyethylene glycol which are connected by acylhydrazone bonds, and the conjugated peptide is loaded in the microsphere.

The microspheres are composed of amphoteric polymers and can self-assemble in water to form microspheres. The hydrophobic portion of the polymer forms the core encapsulating the agent, while the hydrophilic portion forms the shell of the microsphere structure [16]. Acylhydrazone bonds are pH sensitive and can be cleaved at pH <6.8 [17]. According to the local acidic microenvironment of the infarcted area, a lipophilic compound DSPE and a hydrophilic compound PEG can be connected through an acylhydrazone bond (Hyd) to form DSPE-Hyd-PEG (DHP). The inventor verifies that the microspheres have uniform spherical structures, are stable in neutral solution and are depolymerized in acidic solution. Microspheres (DHPM) were loaded with 4APPC to form DHPM _ (4 APPC) with a loading capacity of 32.34 + -7.43 wt%.

In a preferred embodiment of the present invention, the microsphere is cleaved at a pH of less than 6.8. Rupture in an environment with an infarct zone pH <6.8 to release the inner load.

The invention provides an exosome-carrying biological vector, which comprises the pH/H ₂ O ₂ The MMP9 time-sequence response microsphere is characterized in that the periphery of the time-sequence response microsphere is connected with a CD63 aptamer through an acylhydrazone bond, and the CD63 aptamer and an exosome are specifically combined through a CD63 marker on the surface of the exosome (the first mode).

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

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

In an alternative embodiment, the exosome is derived from an adipose mesenchymal stem cell, a bone marrow mesenchymal stem cell, an umbilical cord mesenchymal stem cell, a placental mesenchymal stem cell, a urine-derived stem cell or an endothelial progenitor cell.

CD63 is the most important marker for exosomes and is expressed in large quantities on the surface of exosomes [13]. Aptamers are short oligonucleotide sequences or short polypeptides obtained by in vitro screening, which bind to corresponding ligands with high affinity and strong specificity [18,19]. To modify spark-high Tregs-derived exosomes, the inventors selected the CD63 aptamer as a linker. CD63 aptamers were screened by exponential enrichment of ligand phylogenetic techniques [20], and the top 10 aptamer sequences with the highest score were synthesized and the affinity of each sequence for CD63 protein was verified (Table S). The highest affinity sequence was selected in this study (FIG. S7A). The results of the binding experiments showed that the selected aptamers have strong binding to CD63 (Kd =4.54 ± 0.27 nM) (fig. S7B), fig. S7B is an affinity curve of CD63 aptamers and CD63 protein with increasing concentration.

The pH-sensitive acylhydrazone linkage is set to facilitate the release of exosomes under acidic conditions.

In an alternative embodiment, the CD63 aptamer binds specifically to exosomes derived from SPARC-highly expressed regulatory T cells via a CD63 marker on the surface of the exosomes (second mode).

In an alternative embodiment, the CD63 aptamer binds specifically to exosomes derived from CXCR 2-high expressing and SPARC-high expressing regulatory T cells via a CD63 marker on the surface of the exosomes (third approach). By utilizing the action of chemokines in the first stage of inflammatory response, the exosomes with high CXCR2 expression can be targeted to the infarcted area under the action of chemokines.

The inventors have found that the first, second and third modes can suppress ischemia reperfusion injury, restore cardiac function and prevent fibrosis, and the third mode has a more excellent effect. Namely, the exosome of the regulatory T cell with high CXCR2 expression and SPARC expression has the effects of better inhibiting ischemia reperfusion injury, recovering cardiac function, preventing fibrosis and improving cardiac insufficiency after acute myocardial infarction.

The inventor finds that the exosome derived from the simple Tregs has a certain protective effect on myocardial infarction, and the exosome derived from the spark high-expression Tregs has a strong protective effect on the myocardial infarction. Although repeatedly injecting spark in situ ^high Tregs can improve cardiac function, but survival rates are reduced due to secondary damage to the myocardium. Furthermore, targeted delivery of exosomes and retention of the infarct area is also a challenge. The chemotactic characteristics of the immune cells with high CXCR2 expression are obtained by utilizing the enrichment of chemotactic factors such as CXCL1/2/5 and the like in an infarct area after acute myocardial infarction through a gene editing method. The results indicate that Sparc is highly expressed from CXCR2 ^high Exosomes extracted from tregs can be targeted to infarcted areas quickly and accurately. The inventor also develops a system which can form gel to fix the exosome through in-situ crosslinking in the infarct area after the acute myocardial infarction and can slowly release the exosome under the action of a local microenvironment.

Specifically, referring to FIG. 16, the inventors successfully modified 4A-PEG with a polypeptide specifically hydrolyzed and recognized by MMP9 by adding Co to the solution ²⁺ After that, do not affect the function of the compound, and are in H ₂ O ₂ Forming a gel sample under the action of (3). Meanwhile, the acyl hydrazone bond is used for connecting DSPE and PEG to form DHPM, an aptamer of CD63 is selected to modify the DHPM, the DHPM is combined with and wraps the gel material, and the gel material is combined with Tregs ^Sp_hi_ Exos ^CXCR2_hi Anchoring to form a composite structure. The system successfully targets the infarct area under the action of CXCR2, realizes the rupture of microspheres under acidic condition and utilizes the high H of the infarct 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 traps exosomes. Then, by utilizing the gradual increase of MMP9 in the later stage, the gel is gradually degraded, and the exosome is promoted to be gradually released, so that the effect of protecting acute myocardial infarction by targeting delivery and fixing the exosome as a non-invasive treatment is achieved. The invention provides a new choice for treating acute myocardial infarction.

In an alternative embodiment, the CXCR 2-high expressing and SPARC-high expressing regulatory T cells are gene editing means that allow SPARC-high expressing regulatory T cells to achieve CXCR 2-high expression.

The inventors found Sparc ^high The Treg-derived exosome can provide a new scheme for treating acute myocardial infarction. The surface of an exosome membrane expresses a large amount of CXCR2 through gene engineering editing, the exosome is promoted to rapidly target to an infarct area,

in an alternative embodiment, the sequence of the aptamer is as set forth in SEQ ID NO:1, and the following steps: UUAGCAGUGUACGAGAGUACAAGUUA.

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

In an alternative embodiment, the modification is biotin.

Detectable labels are substances having properties, such as luminescence, color development, radioactivity, etc., which can be observed directly by the naked eye or detected by an instrument, by which qualitative or quantitative detection of the respective target substance can be achieved.

In alternative embodiments, labels that can be detected include, but are not limited to, fluorescent dyes, enzymes that catalyze the development of a substrate, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In actual use, a person skilled in the art can select a suitable marker according to the detection condition or actual need, and the label is not limited to any one of the markers.

In alternative embodiments, fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (e.g., including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxyphoton (FAM), tetrachlorofluorescein (TET), etc. or analogs thereof), rhodamine-based dyes and derivatives thereof (e.g., including, but not limited to, red Rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or analogs thereof), cy-series dyes and derivatives thereof (e.g., including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, etc. or analogs thereof), alexa-series dyes and derivatives thereof (e.g., 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 (e.g., including, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (polymetaxanthin), polymetaxanthin-protein (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 glucose-6-phosphate deoxyenzyme.

In alternative embodiments, the radioactive isotope 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, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium esters and its derivatives, dioxetane and its derivatives, lokaline 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, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

The invention provides a pH/H ₂ O ₂ The preparation method of the MMP9 time-sequence response microsphere comprises the following steps:

mixing the conjugated peptide and microsphere formed by distearoyl phosphatidyl ethanolamine and polyethylene glycol connected by acylhydrazone bond in solvent, centrifuging, and vacuum drying.

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

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

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

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

time-ordered response microspheres were mixed with CD63 aptamer at a concentration of 0.23-0.27mM: mixing 0.008-0.012mM to obtain time sequence response microsphere connected with CD63 aptamer through acylhydrazone bond, and mixing exosome derived from regulatory T cell with the time sequence response microsphere connected with CD63 aptamer. For example, time-ordered response microspheres were mixed with CD63 aptamer at a ratio of 0.257mM: the mixing reaction was carried out at a molar ratio of 0.01 mM.

In an alternative embodiment, before the CD63 aptamer is subjected to the mixing reaction, an ethynyl group is modified on the CD63 aptamer. The azidoacetophenone is modified to facilitate subsequent reaction with the microspheres.

In an alternative embodiment, the mixing mass ratio of exosomes to time-sequentially responsive microspheres linked with CD63 aptamers is 0.5-1.5. Under the above mixing conditions, the combination ratio is more excellent. For example, the mixing mass ratio is 1.

In an alternative embodiment, the mixing is an incubation at 37 ℃. + -. 0.5 ℃ for 3h.

Conjugated peptide, pH/H ₂ O ₂ Application of MMP9 time-sequential response microspheres or exosome-carrying biological carriers in preparation of myocardial infarction repair drugs.

In the above application, pH triggers the release of exosomes, reactive oxygen species (H), of the biological vector ₂ O ₂ ) The oxidation of metallic cobalt ions is initiated to form a gel to immobilize the released exosomes, and then the degradation of the gel is triggered based on the hydrolysis of MMP9 enzyme, slowly releasing the exosomes.

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

The invention provides a medicine for treating myocardial infarction, which comprises the conjugated peptide and the pH/H ₂ O ₂ MMP9 time-ordered response microspheres or the exosome-carrying biological vector described above.

In a preferred embodiment of the application of the present invention, the medicine 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 ampoules.

In an alternative embodiment, the above-described medicament further comprises a combination drug/therapy, which is a combination of the disclosed pharmaceutical compound with at least one of the following drugs/therapies:

chemotherapeutic agents, radiation therapy, photosensitizers, photothermics, immunotherapy, androgen receptor antagonists and function modulators, estrogen receptor antagonists and function modulators, sugar hormone receptor antagonists and function modulators, salt hormone receptor antagonists and function modulators, FXR agonists, antagonists and function modulators, GPR30 agonists, antagonists and function modulators, TGR agonists, antagonists and function modulators, GLP receptor agonists, antagonists and function modulators, FGF receptor agonists, antagonists and function modulators, thyroxine receptor agonists, antagonists and function modulators, sodium-glucose cotransporter 2 inhibitors, dipeptidyl peptidase-4 inhibitors, and TGF receptor agonists, antagonists and function modulators.

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

The invention has the following beneficial effects:

the exosome derived from regulatory T cells based on SPARC high expression has the protection effect on the heart function, and the pH and H in the infarct area ₂ O ₂ And MMP9 level, a conjugated peptide, pH/H ₂ O ₂ MMP9 sequential response microspheres and an exosome-carrying biological carrier.

First, the conjugated peptide has good fluidity, once it is H ₂ O ₂ Triggered, the material exhibits good toughness and strength, which will help it adapt to the beating characteristics of the heart and achieve long-term retention. And once the material is triggered again by MMP9, the toughness and toughness characteristics disappear, and the flow flowsMobility is increased, thereby ensuring release of the loaded reagent. The above-mentioned conjugated peptides are based on H ₂ O ₂ Responsive to design, and Co ²⁺ The 4A-PEG connected in coordination can form hydrogel in situ in the area of the peduncle to fix the exosome. The peptide segment for MMP9 enzyme hydrolysis is arranged in the conjugated peptide, so that the formed hydrogel is gradually degraded and exosomes are slowly released under the action of MMP9 enzyme.

Next, the inventors verified that the above pH/H ₂ O ₂ The MMP9 time-sequence response microspheres are in a uniform spherical structure, are stable in a neutral solution and are depolymerized in an acidic solution. For example, in the environment of an infarcted area, to release the inner load.

Thirdly, the biological carrier carrying the exosome can release the exosome and the hydrogel raw material by pH response and acyl hydrazone bond breakage. Subsequently, using H ₂ O ₂ Mixing Co ²⁺ Oxidation to Co ³⁺ Forming a gel and immobilizing the released exosomes. Finally, upon hydrolysis of MMP9, the gel gradually degrades, releasing exosomes, exerting a sustained and long-term protective effect on the myocardium, improving cardiac function.

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

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

Drawings

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

FIG. 1 is a diagram showing verification of Sparc in Experimental example 1 ^high Treg-derived exosomesFigure of results of body function ((A) use of SD rats (female, 180-230 g) to establish an acute myocardial infarction model following the described surgical procedure, ischemia for 45 minutes and reperfusion for 4 weeks, reperfusion after which purified Tregs were administered by means of local in situ injection ^Sp_lo Exos (0.1 mL; containing 0.1X 10 ⁸ /mL exosomes) or Tregs ^Sp_hi Exos (0.1 mL; containing 0.1X 10 ⁸ /mL exosomes), injected once a day for 7 days, then injected once every 2 days for 7 days, and observed for 2 weeks, PBS group was injected only 1 time; tregs ^Sp_lo Exos stands for Sparc ^low Treg-derived exosomes, tregs ^Sp_hi Exos stands for Sparc ^high Treg-derived exosomes. (B) After 24 hours of reperfusion, the heart was removed and evans blue-TTC staining was performed; each group, n =5. (C, D) measuring the areas of AAR, IF and LV based on the result of (B), and calculating and statistically analyzing the results of IF/AAR and AAR/LV; each group, n =5, one-way anova; IF: infarct size, AAR: hazard zone, LV: the left ventricle. 4 weeks after reperfusion, cardiac function was measured using echocardiography, (E) statistical results of echocardiography were obtained by pulsed doppler and M-mode ultrasound measurements, (F) E/a: the ratio of the ventricular flow velocities at the early and late diastole; (G) LVEF: left ventricular ejection fraction; (H) LVFS: left ventricular rate of shortening; (I) HR: heart rate; each group, n =5; and (4) carrying out one-way analysis of variance. (J, K) after echocardiography, immediately taking the heart and cutting it into 5 μm slices, carrying out (K) massson staining, measuring the area of fibrosis and carrying out (J) statistical analysis; each group, n =5; and (4) performing one-way analysis of variance. (L) in the whole process, monitoring and statistically analyzing the death of the rat to obtain a Kaplan-Meier survival curve; each group, n =20; log-rank (Mantel-Cox). * P is<0.05)；

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

FIG. 3 is a graph showing the result of DHPM (4 APPC) _ Exo characterization;

FIG. 4 is a graph showing the results of experiment in Experimental example 5in which DHPM (4 APPC) _ Exo was confirmed to improve cardiac insufficiency in patients with acute myocardial infarction;

FIG. 5 isolated Sparc ^high Tregs and exosome characterization map of its origin ((A) establishing acute myocardial infarction modelType, 3 days later, the heart was harvested and CD45 was used ⁺ CD4 ⁺ Foxp3 ⁺ Sparc ⁺ Tregs was used as the isolation protocol. Separating CD45 ⁺ CD4 ⁺ Foxp3 ⁺ Sparc ⁺ Tregs and CD45 ⁺ CD4 ⁺ Foxp3 ⁺ Sparc-Tregs were cultured to obtain exosomes, transcribing CXCR2 overexpression plasmids into CD45 ⁺ CD4 ⁺ Foxp3 ⁺ Sparc ⁺ Tregs and obtaining the exosomes from which they are derived. (B) detecting exosome morphology by TEM analysis; (C) Nanoparticle Tracking Analysis (NTA) to detect particle size distribution; (D) measuring zeta potential of the exosomes; (E) western blot detects the expression of exosome markers CD63 and CD9, confirming the efficiency of gene editing by detecting CXCR 2; tregs ^Sp_lo Exos: sparc low expression Tregs-derived exosomes, tregs ^Sp_hi Exos: sparc high expression Tregs-derived exosomes, tregs ^Sp_hi _Exos ^CR2_hi : sparc high expression Tregs-derived exosomes highly expressed from CXCR 2);

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

FIG. 7 is a synthesis scheme for 4APPC and H for 4APP ¹ NMR spectra (4 APP: four-armed polyethylene glycol coupled to MMP9 enzyme conjugate peptide, 4APPC: co based on Metal-ligand interactions ²⁺ Modified 4 APP);

FIG. 8 shows Co ²⁺ Quilt H ₂ O ₂ Oxidation to Co ³⁺ Schematic diagram of (a) and a schematic diagram of a specific peptide fragment of MMP9 enzymatic hydrolysis;

FIG. 9 is a synthesis scheme for intermediate B;

FIG. 10 is a DSPE-Hyd-PEG synthesis route;

FIG. 11 is a graph showing a structure of a folding structure of a CD63 aptamer and a result of affinity assay;

FIG. 12 is a DHPM _ Apt ^CD63 Synthetic roadmaps of (a);

FIG. 13 is a DHPM _ Apt ^CD63 The verification result graph of (1);

fig. 14 (a) according to the results of fig. 4A, areas of AAR and LV were measured, AAR/LV was calculated and statistical analysis was performed, each group, n =5; analyzing single-factor variance; AAR: hazard zone, LV: a left ventricle; (B) HR was detected and analyzed by echocardiography 4 weeks after injection; HR: heart rate; each group, n =5; analyzing the single-factor variance;

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

FIG. 16 shows an exosome-loaded biovectorSchematic of the structure of the body and pH/H ₂ O ₂ Schematic diagram of/MMP 9 time-sequence response.

Detailed Description

Reference will now 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. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. M.j. Goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), academic Press, inc. (Academic Press, inc.), "Handbook of Experimental Immunology" ("D.M.Weir and C.C.Black well"), gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos.), "Current Protocols in Molecular Biology" (F.M.Ausubel et al., 1987), "PCR, polymerase Chain Reaction (PCR: the Polymerase Chain Reaction) (Mullis et al., 1994), and" Current Protocols in Immunology "(blood), each of which is incorporated herein by reference, cold, 1991.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

The materials are as follows:

Boc-H (Trt) GPLGLAGG-OH peptide was purchased from ChinaPeptidesCo.LTD (Shanghai, china); tetraarm polyethylene glycol amine (# A163118, MW2000 Da), 1-hydroxybenzotriazole (HOBT; # H106176 ≥ 97.0%), N-dimethylformamide (DMF; # D112009 ≥ 99.9%), N' -diisopropylcarbodiimide (DIC; # D106162, 98%), N-diisopropylethylamine (DIPEA; # D109321, 99%), trifluoroacetic acid (TFA; # T103295,>99.5%), triisopropylsilane (# T107280, 98%), cobalt dichloride (# C106772, 99.7%), N- (2-hydroxyethyl) -piperazine-N' -ethanesulfonic acid (HEPES; # H109408,. Gtoreq.99.5%), sodium hydroxide (# S291911,60% (w/v)), NH ₂ PEG-COOH (# A163238, MW600 Da), 4-acetylbenzoic acid (# A151401,>98.0%), dichloromethane (# D116153, ≥ 99.9%), N' -dicyclohexylcarbodiimide (DCC; # D106074, 99%), 4-dimethylaminopyridine (DMAP; # D109207, 99%), triethylamine (# T103284, ≧ 99.5%), tert-butyl carbonitride (# B106949, 98%), 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE; # D130471 was set as a control,>97%), succinic anhydride (# S104823, 99%), acetonitrile (# a104439, ≧ 99.9%), copper tetrakis (acetonitrile) hexafluorophosphate (# T115570, 97%), acetylacetone para-azide (# P344177), tetrabutylammonium fluoride solution in tetrahydrofuran (TBAF-THF; # T106821,1.0 MinTHF), 5-ethynyl-2' -deoxyuridine (EdU; # E131265,. Gtoreq.98%), cy5.5 (# C266425), fluorescein isothiocyanate (# F272903,. Gtoreq.95%), hydrogen peroxide (# H112517,30wt% ₂ O) was purchased from Aladdin. Methylene trichloride (# 288306, ≧ 99%), acetone (# 48358), triethylamine acetate buffer (TEAA; #90358, 1.0M), biotin (# B4501, ≧ 99%) were sufficient from SigmaAldric. 4-amino-DL-phenylalanine (# 235606) was purchased from J&KScientific。

Example 1

This example provides a conjugated peptide and a method for preparing the same.

Reference is made to the methods described in the following documents:

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

4-ArmPEGAMINe (5.0 g,2.0mmol NH) ₂ end-group), boc-H (Trt) GPLGLAGG-OH (2.0 g,4.0 mmol) and HOBTHOBT (0.54g, 4.0 mmol) were dissolved in DMF solution (15 mL), followed by the addition of DIPEA (1.07mL, 6.0 mmol) and DIC (0.62mL, 4.0 mmol), and the mixture was reacted at room temperature for 24 hours. The crude precipitate was washed 3 times with cold ether and the resulting white powder precipitate was dried in vacuo and then further dissolved in a cleavage solution consisting of TFA (95 mL), triisopropylsilane (2.5 mL) and water (2.5 mL) and stirring was continued for 3 hours to remove the protecting group. The solvent was removed under reduced pressure, the product was further dissolved in DMF in the appropriate amount and purified with cold diethyl ether (3 ×) to give 4APP. To further synthesize 4APPC, GOX (0.4 mg/mL; # G8030,. Gtoreq.300U/mg, solarbio), 4APP (100 mg/mL,10 mM) and CoCl2 (13.3 mM) were mixed in HEPES buffer (pH 7.0), the solution pH was adjusted to 7.0 with NaOH, and after 2 hours of reaction at room temperature, co was obtained ²⁺ The hydrogel was crosslinked and the product centrifuged (. Times.1500 g) for 30 min and then dried under vacuum to give 4APPC as the final product. 4APPC by means of a nuclear magnetic resonance spectrometer (Bruker Avance400 spectrometer at 400 MHz) ¹ H spectrum detection was confirmed.

The synthetic structure of 4APPC is shown in FIG. 7A. The 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-sequence response microsphere (DHPM (4 APPC)) and a preparation method thereof.

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

synthesizing DSPE-Hyd-PEG:

4-Acetylbenzoic acid (0.5 mM) was dissolved in a dichloromethane solution (2 mL), and DCC (1.0 mM) and DMAP (1.0 mM) were added to react at room temperature for 6 hours; NH2-PEG-COOH (0.25 mM) was added, along with triethylamine (80. Mu.L). The mixture was allowed to retract for 6 hours with continued gentle shaking at room temperature, the reaction solution was filtered and evaporated, and the residue was dissolved in an appropriate amount of ultrapure water and dialyzed (1000 Da; # YA1035, solambio; 500Da, # YA1069, solambio) at room temperature for 48 hours, followed by lyophilization to give product A. Mixing the product A (0.5 mM) and tert-butyl carbamate (0.25 mM), 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 a product B.

DSPE (0.2 mM) was dissolved in methylene trichloride (2 mL), succinic anhydride (0.2 mM) was dissolved in DMSO (0.5 mL), and then the solution was added dropwise to the DSPE solution, and the mixture was reacted for 24 hours with exclusion of light. After completion of the reaction, acetone (45 mL) was added, and the reaction was further carried out overnight at-20 ℃ and centrifuged (15000 rpm) for 15 minutes to remove the supernatant, and the solid was collected as product C. The product C (0.5 mM), DCC (1.0 mM) and DMAP (1.0 mM) were mixed in a dichloromethane solution (2 mL) and reacted at room temperature for 6 hours; to the reaction mixture were added tert-butyl carbamate (0.5 mM) and triethylamine (80 μ L), and 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) to obtain product D. Further, the product D (0.5 mM) was dissolved in dichloromethane (2 mL), TFA (2 mL) was added, the reaction was stirred at room temperature for 2 hours, the reaction was stopped, the reaction solvent was evaporated, and the product E was obtained by chromatography on a silica gel column (eluent dichloromethane: methanol = 10.

Mixing product B (0.3 mM) and product E (0.3 mM) in dichloromethane (2 mL), adding TFA (100. Mu.L), stirring at room temperature for 8 hours, stopping the reaction and evaporating the reaction solvent, dissolving the residue in an appropriate amount of ultra-pure water, dialyzing at room temperature (1000 Da; # YA1035, solarbio) for 48 hours, lyophilizing to obtain DSPE-Hyd-PEG, and using a nuclear magnetic resonance spectrometer (400 MHz of Bruker Avance 400) for the ¹ And H spectrum detection.

DSPE-Hyd-PEG (100 mg) and 4APPC (50 mg) were dissolved in dichloromethane (10 mL) with constant low speed stirring while slowly adding H ₂ O (4.3 mL) was added by syringe pump at a rate of 0.5 mL/hour until the water concentration was 30% v/v, stirring was continued for 60 hours, and then H was continuously added slowly at a rate of 2 mL/hour ₂ O(5.7mL)，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 the same procedure is performed without adding 4APPC, the final product is DHPM. After DHPM (4 APPC) is formed, the supernatant is detected by an ultraviolet-visible spectrophotometer, and the loading rate of the 4APPC is calculated according to beer's law. Loading (wt%) = (4 APPC total weight-remaining 4APPC weight)/(DHPM weight) × 100%.

Example 3

This example provides a method for linking CD63 aptamer through acylhydrazone bond at the periphery of a time-sequential response microsphere (DHPM _ Apt) ^CD63 Preparation).

The screened CD63 Aptamer (shown by reference SEQ ID NO: 1) is modified by ethynyl (Aptamer) ^CD63 CCH), synthesized by standard RNA synthesis procedure based on solid phase synthesis, i.e. terminal deoxyuridine was replaced by EdU, nucleotide chains were cleaved from solid support, controlled Pore Glass (CPG) support (2 mg) was blown dry with nitrogen, acetonitrile (0.5 mL) and tetrakis (acetonitrile) copper (I) hexafluorophosphate (20 mM) were added followed by p-azidoacetophenone (1 μ M), shaking continuously for 16 hours at room temperature, centrifugation (3000 rpm) for 10 minutes at room temperature and discarding supernatant, the residual solid phase was washed 3 times with 1mL acetonitrile. Then, a mixture (2 mL) of ethanol and concentrated aqueous ammonia (1). Extracting the solution twice with chloroform (0.5 mL), adding TEAA buffer solution (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 the theoretical molecular weight [ M + Na + ] of the product F by MALDI-TOF]=10092, MALDI-TOF molecular weight detected 10094.317.

Similar to the previous procedure, DHPM (0.5 mM) was dissolved in dichloromethane (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 evaporated, the residue was washed with ultrapure water 3 times, centrifuged (1500 rpm) at room temperature for 5 minutes, and dried in vacuo to give product G.

In addition, as described above, an acylhydrazone bond may be formed between the product F and the 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), 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, the reaction was stirred slightly at room temperature for 24 hours, then centrifuged (3000 rpm) at room temperature for 10 minutes, washed 3 times with ultrapure water (2 mL), and dried under vacuum to give DHPM _ Apt ^CD63 。

Example 4

This example provides a method for preparing an exosome-loaded biological vector, which comprises subjecting the DHPM _ Apt prepared in example 3 to ^CD63 DHPM (4 APPC) _ Exo was prepared by incubation in admixture with exosomes.

Isolating cells

Cells infiltrating the heart were isolated according to previously reported methods (Xian, luY, guM, liN, liuM, jianoJ, zhuZ, liJ, liD, tangT, lvB, nieS, zhangM, liaoM, liaoY, yangX, chengX, queuing of RegulatoryTCellsinHeartPtitiates Cardiac protection information M Myocardialination.circulation.2020 Nov17;142 (20): 1956-1973.). Briefly, an acute myocardial infarction model was established 3 days later, heart tissue was harvested, minced, and the tissue mass was treated with collagenase II (1 mg/mL) in HEPES buffer (# 17101015, gibco ^TM ) Digestion, gentle rotation at 37 ℃ for 1.5h, cell suspension with cell filter (40 μm) (# CL FIG. 831750, corning falcon ^TM ) The mixture is filtered and then is filtered,

PM400 (# F4375, sigma-Aldrich) density centrifugation purified cardiac mononuclear cells. Cardiac mononuclear cells were collected using CD4MicroBeads (# 130-090-319, miltenyi Biotec), isolated cells were stained using anti-CD 45 (# ab10558, abcam), anti-CD 4 (# ab6413, abcam), anti-Foxp 3 (ab 215206, abcam) and anti-Sparc (# ab290647, abcam), and finally, CD45 was stained using a flow cytometer CSA faria instrument (BD Bioscience, USA) ⁺ CD4 ⁺ Foxp3 ⁺ Sparc ⁺ Tregs were sorted and analyzed using FlowJo software.

Cell culture and lentivirus transfection

Sorted Tregs were cultured in RPMI-1640 complete medium (10% FBS) containing CD3 ε antibody (5. Mu.g/mL; #100340, biolegend) and anti-CD 28 antibody (2.5. Mu.g/mL; #102116, biolegend) at 37 ℃ and 5% CO2. After 24 hours of culture, a CXCR 2-highly expressed Crispr-cas9 lentivirus-packaged plasmid (GenePharma; sgRNA: GCATAGTCTGAGAGATTCTTGCT) was transfected and cultured for 48 hours.

Exosome purification and characterization

Exosomes were purified by continuous centrifugation. Briefly, cell supernatants were collected and centrifuged (300 Xg) at 4 ℃ for 10 minutes, the supernatant was retained and centrifuged (10000 Xg) at 4 ℃ for 30 minutes again, then the supernatant was collected again, centrifuged (140000 Xg) at 4 ℃ for 90 minutes continuously, the supernatant was removed, resuspended and washed with an appropriate volume of PBS, centrifuged (140000 Xg) at 4 ℃ for 90 minutes to collect the pellet, and frozen at-80 ℃ until use. Use of Pierce ^TM Rapid Gold BCA Protein Assay(#A53226,ThermoScientific ^TM ) And detecting the total protein concentration. The particle size distribution and particle concentration were measured using an NTA nanosize Analyzer Nanosight LM10 (Malvern Instruments). In addition, the morphology of the exosomes was observed using Transmission Electron Microscopy (TEM), the exosome suspension was placed on a carbon grid and stained with uranyl acetate (2%), and the morphology was observed by H-7000FATEM at 80-kV. The exosome markers CD9 and CD63 were detected by western blot.

Spark with high expression of CXCR2 ^high Tregs-derived exosomes and DHPM _ Apt prepared in example 3 ^CD63 After mixing at 37 ℃ for 3 hours, 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 And (3) binding with an exosome, loading the exosome with a fluorescent molecule FITC, loading DHPM with Cy5.5, finally forming DHPM (Cy5.5) _ Exo (FITC), and observing the fluorescence distribution of the DHPM (Cy5.5) _ Exo (FITC) through a fluorescence microscope. Detection of DHPM _ Apt by western blot ^CD63 With exosomesThe binding rate was determined by detecting CD63 by SDS-PAGE after lysis of bound exosomes in DHPM _ Exo with RIPA lysate (# P0013B, beyotime) using the optimal binding amount ratio against total exosomes. Detection of DHPM _ Exo or DHPM (4 APPC) _ Exo (at DHPM _ Apt) by western blot ^CD63 In the forming process, the DHPM is loaded with 4APPC to form DHPM (4 APPC) _ Apt ^CD63 ) To pH<6.8、H ₂ O ₂ Or MMP9 enzyme (# P01795, solarbio), released exosomes were validated by CD63 detection and quantified with NTA.

Experimental example 1

This example examines Sparc obtained by purification in example 4 ^high Treg-derived exosomes, and the functions thereof are verified.

The experimental method is as follows:

(1) And (5) constructing an acute myocardial infarction model.

An acute myocardial infarction model was established according to a previously reported method (ChengP, hanH, chenF, chengL, maC, huang H, chenC, liH, caiH, huang H, liG, taoJ. Aminoj. Aminothionation of acutematic tissue and fertilidinocagesloaded with ALKBH5inhior. Acta Biomater.2022Mar1; 140. Briefly, all animal-related procedures were approved by the animal protection and use committee using SPF-grade SD rats (female, 180-230 g) for modeling. SD rats are anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg), are intubated through a trachea, are connected with a small animal respirator to maintain the respiration of the mice, and are monitored and evaluated for myocardial ischemia through Electrocardiogram (ECG). Open chest surgery was performed in the fourth intercostal region on the left to expose the heart, and the left anterior descending coronary artery was ligated with sterile 6-0 thread, leaving a knot 2-3 mm distal to the LCA origin. Myocardial ischemia is prompted when T wave inversion and ST segment elevation appear on the electrocardiogram, and knots are loosened after 45min of ischemia to perform reperfusion. Meanwhile, different treatment strategies are adopted, and different detections are carried out at different time points. The same experimental procedure was used for the sham operation group except for the coronary artery ligation.

(2) Transmission Electron Microscope (TEM)

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

(3) Particle size distribution and zeta potential detection

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

(4)Western blot

Exosomes or other samples were lysed with RIPA lysis buffer (# P0013B, beyotime) on ice for 30 min, then centrifuged (10000 × g) at 4 ℃ for 15 min, and western blot analysis was performed using the supernatant. The purified proteins were separated on a 10% -15% SDS-PAGE gel, transferred to PVDF membrane, incubated for 1 hour at 37 ℃ in skimmed milk (5%) and then overnight at 4 ℃ with primary antibodies as follows: anti-CXCR 2 (# ab65968, abcam), anti-CD 63 (# PA5-92370, invitrogen), anti-CD 9 (# PA5-85955, invitrogen) and anti-beta-actin (# ab8226, abcam). The membrane was then washed 3 times with PBST (10 minutes each), incubated with secondary anti-mouse IgG (# ab6789, abcam) or anti-rabbit IgG (# ab6721, abcam) for 2 hours at 37 ℃, washed 3 times with PBST (10 minutes each), chemiluminescent with BeyoECLMoon (# P0018FS, beyotime), and bands were obtained by the FluorChemE data System (ProteinSimple, CA, USA).

The above test results show that Sparc ^high Tregs play an important protective role in tissue repair after acute myocardial infarction [2]. To detect spark ^high Influence of Treg-derived exosomes on acute myocardial infarction, tregs are separated into Sparc by a flow cytometric separator ^high And spark ^low (FIG. 5A) and collection of exosomes of different cell origin by ultracentrifugation [13]]. Transmission electron microscopy showed that the purified exosomes maintained good integrity (FIG. 5B), as detected by nanoparticle tracking analysis and dynamic light scattering, sparc ^high And spark ^low There was no significant difference in the particle size distribution and zeta potential of the derived exosomes (fig. 5C and 5D)). Western blot detection of specific markers CD9 and CD63 of exosomes further confirms that exosomes are successfully separated from Sparc ^high Tregs and spark ^low Tregs (fig. 5E).

The inventors determined Sparc by constructing an acute myocardial infarction model of Sprague-Dawley (SD) rats ^high And spark ^low Influence of the source exosomes on cardiac function. Exosomes were injected in situ into the myocardium of SD rats 2 times a day for the first week and 1 time a day for the second week (fig. 1A). 24 hours after the first injection, the heart was harvested and sectioned, stained with evans blue, and then immersed in triphenyltetrazolium chloride. The results show that in-situ injection of Tregs-derived exosomes can effectively inhibit ischemia-reperfusion injury, while Sparc ^high The effect of Treg-derived exosomes was more pronounced (fig. 1B-1D). After four weeks, the inventor detected the cardiac function condition by echocardiography, and the result shows Sparc ^high Treg-derived exosomes were effective in promoting restoration of cardiac function, including E/a ratio, left ventricular shortening rate and left ventricular ejection fraction (fig. 1E-1I). In addition, further detection of myocardial fibrosis by Masson staining indicates Sparc ^high Treg-derived exosomes effectively inhibited fibrotic scarring (fig. 1J and 1K). The above results indicate that spark ^high And spark ^low The obtained exosomes can improve the cardiac function after acute myocardial infarction. However, in situ injection also resulted in a sustained decrease in survival rate, eventually approaching 30% (fig. 1L).

Experimental example 2

This example demonstrates that the time-ordered responsive microspheres and exosome-loaded biological vectors prepared in examples 2-4 have an exosome delivery function and are immobilized in an infarct region, and demonstrates that the conjugated peptide prepared in example 1 has good fluidity and is once bound by H ₂ O ₂ Triggered, the material exhibits good toughness and strength, which will help it adapt to the beating characteristics of the heart and achieve long-term retention. And once the material is again triggered by MMP9, the toughness and elastic characteristics disappear and the fluidity increases, thus ensuring the release of the loaded agent.

In the early stage of acute myocardial infarction, a large number of CXCR2 high-expression immune cellsCells accumulate in the infarct area under the action of chemokines [ 9]]. To ensure that exosomes were able to target infarct regions, the inventors used the CRISPR/Cas9 system pair Sparc ^high Tregs undergo gene editing to increase CXCR2 expression on the surface of exosome membranes. And this was not significantly different from the characteristics of exosomes obtained in unedited cells (fig. 5B-1E). The inventors loaded Fluorescein Isothiocyanate (FITC) into exosomes and injected it into veins of SD rats after acute myocardial infarction. Spark with high CXCR2 expression as shown by bioluminescence imaging ^high Treg-derived exosomes can be rapidly targeted to the heart (fig. 6A) and are mainly concentrated in the infarct zone (fig. 6B). It was found that the number of exosomes in the infarct area peaked after about 2.5 hours and then gradually decreased upon a single injection of exosomes (fig. 6C and 6D). If exosomes were injected for 7 consecutive days, the number of exosomes that accumulated in the infarct zone would gradually decrease over time (fig. 6E-6H). The results indicate that CXCR2 can efficiently direct exosomes to the infarct area, but the exosomes do not have long residence time in the infarct area and the targeting ability decreases with time. This is probably due to the washout of blood flow and the gradual decrease in CXCR2 ligand over time, resulting in exosomes that do not remain in the infarct zone for long periods of time.

To solve this problem, the inventors designed a hydrogel-microsphere-exosome complex system, i.e., DHP microspheres (DHPM) (4 APPC) _ Exo (refer to the exosome-carrying bio-vector provided in example 4). Previous studies have shown that the amino group and Co in histidine are present in aqueous histidine-modified 4A-PEG solution ²⁺ Coordinate bonds may be formed. At H ₂ O ₂ By the action of (C), co ²⁺ Oxidation to Co ³⁺ While the 4A-PEG aqueous solution changes from liquid to gel-like [14 ]]. H produced in the second stage of the inflammatory reaction ₂ O ₂ And MMP9 formed in the fourth stage is continuously accumulated in the infarct zone [10-12 ]]MMP9 can specifically recognize and hydrolyze the peptide fragment "GALLGLP" [15]. The change of local microenvironment at different stages after acute myocardial infarction is utilized to ensure that the product has the local microenvironment H ₂ O ₂ The property of forming a gel under the action of MMP9 and hydrolyzing 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, conjugation to Co based on metal-ligand interactions ²⁺ Finally, 4A-PEG conjugated peptide (4 APPC) is 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 transform H ₂ O ₂ Adding into 4APPC aqueous solution to determine 4APPC to H ₂ O ₂ And MMP 9. 4APPC (40 mM) was dissolved in water and H was added ₂ O ₂ (1.0 wt%) triggered hydrogel formation and the rheological properties of the conjugated peptides were examined and their degradation triggered by the addition of MMP9 (0.05. Mu.g/mL).

Using HAAKE ^TM MARS 40Rheometer(Thermo Scientific ^TM ) Rheological properties were tested with parallel plate geometry (25mm diameter,0.3mm gap); fixed strain (stress) frequency test: 5% strain, 0.628-100 rad/s,37 deg.C, pH7.0; yield stress test: oscillating mode, 37 ℃, ph7.0; modulus test: shaking mode, 6.28rad/s,37 ℃,0-1 hours detection, pH7.0.4APPC solution (40 mM) to which H was added ₂ O ₂ (1 wt%) formed a gel, and sufficient MMP9 was added to degrade the gel. In the gel formation time test, H is added ₂ O ₂ After addition of the 4APPC solution, G 'and G' were tested and recorded.

Fig. 2 (a) shows physical forms under different trigger conditions. And (B) frequency scanning measurement. And (C) dynamic time scanning measurement. (D) yield stress test in vibration mode.

The results show that ₂ O ₂ Oxidation (fig. 8A) the liquid becomes gel-like (fig. 2A). However, once MMP9 was added, the solid became liquid again (fig. 2A), apparently by hydrolysis of the polypeptide chain (fig. 8B). The inventors evaluated the mechanical properties using a frequency sweep test, and the results showed that 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 The time to trigger gelation. When H is present ₂ O ₂ After addition of 4APPC, an intersection between the storage modulus (G ') and the loss modulus (G') was observed at 341 secondsCross point, which indicates H ₂ O ₂ Triggered 4 APPCs can form gels. In addition, the stress strain test results 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 show that 4APPC has good flowability once it is H ₂ O ₂ Triggered, the material exhibits good toughness and strength, which will help it adapt to the beating characteristics of the heart and achieve long-term retention. And once the material is again triggered by MMP9, the toughness and toughness characteristics disappear and the fluidity increases, thus ensuring the release of the loaded agent.

The following experiment demonstrates that DHPM remains stable in neutral solution and depolymerizes in acidic solution.

The amphiphilic copolymers can self-assemble in water and form microspheres. The hydrophobic portion of the polymer forms the core encapsulating the agent, while the hydrophilic portion forms the shell of the microsphere structure [16]. Hydrazone bonds are pH sensitive and can be cleaved at pH <6.8 [17]. According to the local acidic microenvironment of the infarcted area, a lipophilic compound DSPE and a 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 resulting structure of DHP is shown in fig. 9A and 11A.

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

DHPM was dissolved in a solution with 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 examined by TEM, nanoparticle size and zeta potential analyzers.

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 confirmed a uniform distribution of DHPM (fig. 2F and 2G). DHPM was added to solutions at different pH values to test their stability. The results show that DHPM remains stable in neutral solution and depolymerizes in acidic solution (fig. 2E-2G). In addition, loading 4APPC with DHPM resulted in DHPM _ (4 APPC) with a loading capacity of 32.34. + -. 7.43wt%. DHPM (4 APPC) can also be rapidly depolymerized in acidic solution (pH < 6.8) (fig. 2H).

Experimental example 3

This experimental example verifies that DHPM _ Apt is used in the above-described embodiment ^CD63 The successful synthesis of.

CD63, the most important marker for exosomes, is expressed in large amounts on the surface of exosomes [13]. Aptamers are short oligonucleotide sequences or short polypeptides obtained by in vitro screening, which bind to the corresponding ligand with high affinity and strong specificity [18,19]]. To modify spark-high Tregs-derived exosomes, the inventors selected the CD63 aptamer as a linker. Ligand System by exponential enrichment [20]The CD63 aptamers were screened, the top 10 aptamer sequences with the highest score were synthesized, and the affinity of each sequence to the CD63 protein was evaluated (table S). The highest affinity sequence was selected in this study (FIG. 11A). The results of the binding experiments showed that the selected aptamers have strong binding to CD63 (Kd =4.54 ± 0.27 nM) (fig. 11B). CD63 aptamers were ethynyl modified (FIG. 12A) and then combined with DHPM to form DHPM _ Apt ^CD63 Which 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 The synthesis is successful, and DHPM loads fluorescent molecules Cy5.5 to form DHPM (Cy5.5) _ Apt by modifying CD63 aptamer through biotin ^CD63 Biotin, followed by mixing of microspheres of different composition with Dynabeads ^TM M-450Streptavidin (5 uL per 1mL solution; #2850000005, thermo Fisher Scientific), incubated at room temperature for 1 hour with gentle shaking, centrifuged (1000 rpm) at room temperature for 10 minutes, observed for color change and photographed. Another strategy is to modify CD63 aptamers by Cy5.5 to form DHPM _ Apt ^CD63 Cy5.5, detection of DHPM _ Apt by flow cytometry ^CD63 -fluorescence intensity of Cy5.5. Both strategies confirm DHPM _ Apt ^CD63 The successful synthesis of the compound.

The final structure is shown in fig. 13A. As a result, the experiment of combining biotin with the CD63 aptamer confirms that the CD63 aptamer is successfully modified on the surface of DHPM. The inventors loaded Cy5.5 with DHPM and incubated with streptavidin magnetic beads that specifically bind biotin, followed by centrifugation, and liquid fluorescence stratification phenomenon confirmed successful modification of the CD63 aptamer on the DHPM surface (FIG. 13B). While cy5.5 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).

Watch S

Experimental example 4

Design experiments in this experimental example show that DHPM (4 APPC) _ Exo effectively targets infarcted areas.

To examine the ability to target to the heart and infarcted areas, the inventors performed optical bioluminescence imaging and fluorescence microscopy. Briefly, once the acute myocardial infarction model was established and different treatment regimens were administered, animals were sacrificed by anesthesia after different times and optical bioluminescence imaging analysis of harvested heart, lungs, liver, kidneys and spleen was performed by Xenogen in vivo imaging system (IVIS, calipers life sciences, USA). Meanwhile, the heart was frozen and cut into 20 μm sections, the sections were observed by a panoramic Tissue section high resolution fluorescence scanning imaging system (THUNDER Imager Tissue, leica), and the fluorescence intensity was statistically analyzed.

DHPM (4 APPC) _ Apt ^CD63 And Tregs ^Sp_hi _Exos ^CXCR2_hi (the spark high-expression Tregs-derived exosome transformed into the CXCR2 over-expression plasmid) to obtain the DHPM (4 APPC) _ Exo with the composite structure. The structure of DHPM (4 APPC) _ Exo was obtained by transmission electron microscopy (FIG. 3A). It can be seen that the bubble-like structure clearly adheres to DHPM (4 APPC) _ Apt ^CD63 Thus, the successful synthesis of DHPM (4 APPC) _ Exo was confirmed. In addition, DHPM _ Apt was added before culture ^CD63 Cy5.5 was loaded and Tregs were combined ^Sp_hi _Exos ^CXCR2_hi FITC was loaded. Successful combination of DHPM _ Exo was further confirmed using fluorescence confocal microscopyTo (fig. 3B).

To determine the optimal binding ratio, tregs were assigned ^Sp_hi _Exos ^CXCR2_hi And DHPM (4 APPC) _ Apt ^CD63 Co-cultured at different ratios. The optimal binding ratio was determined to be 1 by Western blot analysis of CD63 (fig. 3C).

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

FIG. 3E measurement of exosome amount released by DHPM (4 APPC) _ Exo by NTA after incubation in solution at pH <6.8 or 7.2. As shown in fig. 3E, a small amount of exosomes were released at pH 7.2 and a large amount at pH < 6.8.

To verify H ₂ O ₂ And MMP 9-triggered exosome release, DHPM (4 APPC) _ Exo was mixed with pH prior to MMP9 addition<6.8 and contain H ₂ O ₂ The solution culture of (4). In particular, at (pH)<6.8+1.0wt％H ₂ O ₂ ) Or ((pH)<6.8+1.0wt％H ₂ O ₂ ) +0.05 μ G/mL MMP 9), released exosomes were quantified by western blot of fig. 3 (F) and NTA assay of fig. 3 (G), and supernatants were collected for exosome assay. Expression of CD63 in the supernatant was detected by Western blot and found at pH<6.8 time release of exosomes, which are subsequently substituted by H ₂ O ₂ Is fixed and then released again by the trigger of MMP 9. This was also confirmed by quantitative detection of exosomes (fig. 3E and 3G).

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

In all of the experimental examples, SD rats (female, 180-230 g) were used to establish acute myocardial infarction according to the aforementioned methodModel, once blood supply in the ischemic area was restored, DHPM (FITC) (0.2mL, 0.14X 10) was injected via tail vein ⁸ Per mL; containing 0.02. Mu. Mol FITC or DHPM (FITC). Multidot.Exo (0.2mL, 0.2X 10 ⁸ Per mL; comprises 0.1 × 10 ⁸ Per mL exosomes and 0.02 μmol FITC), 2 hours later the heart, liver, spleen, lung and kidney were extracted for optical bioluminescence imaging analysis (n =3 per group), the heart was removed and cut into 20 μm sections for fluorescence distribution visualization. FITC forms DHPM (4 APPC) _ Exo (FITC) through exosome loading, and DHPM (4 APPC) _ Exo (FITC) (0.2mL, 0.2X 10X-phase injection via tail vein once blood supply in ischemic area is recovered in acute myocardial infarction model ⁸ Per mL; contains 2.4mM 4APPC, 0.1X 10 ⁸ Per mL exosomes and 0.02 μmol FITC), 3 (n = 3), 5 (n = 3) and 7 days (n = 3) post injection, major organs were extracted for optical bioluminescence imaging determination and analysis of fluorescence distribution, hearts were cut into 20 μm sections, and the area of infarct and distal fluorescence intensity were observed and analyzed.

Optical bioluminescence imaging and myocardial slice fluorescence imaging indicated that DHPM (4 APPC) _ Exo efficiently targeted infarct areas (fig. 3H and 3I) and resided for more than 7 days (fig. 3J-3M).

Experimental example 5

Design experiments in this experimental example show that DHPM (4 APPC) _ Exo can improve cardiac insufficiency after acute myocardial infarction.

The experimental method is as follows:

evans blue-triphenyltetrazolium chloride (TTC) dye

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 At Risk (AAR) was stained by right jugular vein injection of erwinia blue dye solution (1%, 0.3 ml). Once the heart turns blue, the heart is 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%) at 37 ℃ for 15 minutes, fixed in formalin (10%) overnight, and then photographed using a digital camera. The red area indicates AAR, the white area indicates infarction, and the IF/AAR and AAR/LV values were calculated.

Echocardiogram

MyLabSat (Esaote) was equipped with a phased array probe (SP 3630) for echocardiography. The rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg) and placed on a heating plate at a constant temperature of 37 ℃. And the medical ultrasonic coupling agent is adopted to ensure coupling and ultrasonic transmission. Detecting the diameter of the posterior wall, the diameter of the ventricular septum, the length of the left ventricle and the diameter of the left ventricle in diastole and systole by adopting M-type and pulse wave Doppler ultrasonic cardiography; heart rate, early diastolic filling peak of the left ventricle (peak E) and late diastolic filling peak (peak a) were detected simultaneously. Obtaining an ultrasonic image, and calculating various values of different indexes.

Masson staining

Massson staining was performed using massson's trichrome staining kit (# G1340, solarbio), images were observed and acquired by AperioCS2 instrument (laika biosystems limited), infarct size was measured by ImageJ software, and infarct rate was calculated as infarct size/left ventricular area × 100%, according to the manufacturer's instructions.

Survival monitoring

Rats were monitored daily for mortality after surgery, for peripheral and intrathoracic thrombosis, and infarcted ventricular wall perforation, all 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 (fig. 4A, 4B and 14A), echocardiography (fig. 4C-4F and 14B) and Masson staining (fig. 4G and 4H) were performed.

An acute myocardial infarction model was established using SD rats (female, 180-230 g) according to the above procedure, and once blood supply in the ischemic area was restored, PBS (0.2 mL), DHPM (4 APPC) (0.2mL, 0.2X 10) were injected via tail vein ⁸ Per mL; containing 2.4mM 4 APPC), exos (0.2 mL, containing 0.1X 10 ⁸ /mL exosome) or DHPM (4 APPC) _ Exo (0.2mL, 0.2X 10 ⁸ Per mL; contains 2.4mM 4APPC, 0.1X 10 ⁸ /mL exosomes).

In FIG. 4, A is a graph showing the results of harvesting hearts and staining with Evans blue-TTC 24 hours after injection; per group, n =5. (B) Calculating IF/AAR results and performing statistical analysis for measuring AAR and IF areas; each group, n =5; analyzing single-factor variance; IF: infarct size, AAR: a hazardous area. 4 weeks after injection, performing echocardiography, (C) obtaining pulse wave doppler and M-mode images, obtaining statistical results of echocardiography, (D) E/a: ratio of ventricular flow velocities at early and late diastole, (E) LVEF: left ventricular ejection fraction, (F) LVFS: left ventricular shortening score; each group, n =5; and (4) performing one-way analysis of variance. (G, H) immediately after echocardiography the heart was removed and cut into 5 μm sections, (G) Masson staining for measurement of fibrotic regions, (H) statistical analysis; each group, n =5; one-way analysis of variance. (I) In the whole process, monitoring and statistically analyzing the death of the rat 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. It is noted that the effect of DHPM (4 APPC) _ Exo is better. At the same time, DHPM (4 APPC) _ Exo injection increased survival to around 95%, significantly higher than multiple exosome injections in situ (fig. 4I and 1L).

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

All data are expressed as mean ± standard deviation. Statistical analysis was performed using the T test and the non-parametric Mann-Whitney rank sum test for inter-sample differences. One-way anova and Bonferroni post hoc tests were also used for specific experimental analyses. Differences in animal survival were analyzed by the Kaplan-Meier method. A threshold value 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 indicate 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 induces an inflammatory response that results in the formation of collagen-rich scars, replacing necrotic tissue to prevent cardiac rupture [21]. Following acute myocardial infarction, T cells are involved in inflammation and tissue repair, and Tregs mediate organ-specific regeneration [22]. More importantly, following acute myocardial infarction, spark ^high Treg infiltration into myocardium can increase collagen content, prevent cardiac rupture, and increase survival rate [23]. Thus, spark ^high Tregs can potentially promote cardiovascular tissue repair after AMI by terminating the pro-inflammatory phase and initiating an anti-inflammatory or repair phase. However, cell expansion cannot be achieved in a short time. In addition, the 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]. Therefore, a new class of therapeutic regimens that avoid the drawbacks of cell therapy is urgently needed.

Exosome is a vesicle secreted by cells, has a phospholipid bilayer, has a diameter of 50-150nm [25], carries and transmits key signal molecules, forms a new intercellular information transmission system, influences the physiological state of the cells, and is 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), micrornas (mirnas), and other non-coding RNAs. [27]. Research has shown that microRNAs in exosomes play a key regulatory role in cardiovascular diseases [28], exosomes from immune cells can promote immune response and inflammation of various cardiovascular diseases, and different immune cells have different functions [29]. However, no study has reported the role of the exosomes secreted by Tregs in the cardiovascular system.

To investigate the role of the Tregs of a particular group in myocardial infarction, spark was studied in SD rats ^high The role of Treg-derived exosomes in acute myocardial infarction. The result shows that the exosome of the simple Tregs source has a certain protection effect on myocardial infarction, and the Tregs with high expression of spark come fromThe exogenous exosome has strong protective effect on myocardial infarction. Although repeatedly injecting spark in situ ^high Tregs can improve cardiac function, but survival rates are reduced due to secondary damage to the myocardium. Furthermore, targeted delivery of exosomes and retention of the infarct area is also a challenge. Thus, there is a need for a non-invasive treatment regimen.

Researchers have been shown to target their tissues and even cells by designing specific ligands through various modifications on their surfaces [30 ]]. The currently available modification methods are mainly divided 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 engineering editing is most widely applied due to the advantages of strong editability, low damage, high stability and the like. In the research, the high expression of CXCL (chemokine receptor) related chemokines in an infarct area after acute myocardial infarction is utilized, and the immune cells with high CXCR2 expression are obtained by a gene editing method and have good chemotactic properties. The results show that Sparc is highly expressed from CXCR2 ^high Exosomes extracted from tregs can be targeted to infarcted areas quickly and accurately.

Hydrogels have been used to improve the retention of exosomes in the infarct area. Hydrogels are physically or chemically crosslinked three-dimensional hydrophilic polymer networks that can adsorb large amounts of a target agent without undergoing a dissolution process. In regenerative medicine, hydrogels can serve as scaffolds, barriers, drug delivery systems, and cell encapsulation matrices [32 ]]. Studies have shown that cells or bioactive molecules bound to hydrogels can retain their structure and function for longer periods of time than cells or bioactive molecules not containing hydrogels [33]. Therefore, a system which can form gel to fix the exosome through in-situ crosslinking in an infarct area after the acute myocardial infarction and can slowly release the exosome under the action of a local microenvironment is urgently needed to be developed. There are many current treatment regimens for acute myocardial infarction using 4-Arm-polyethylene glycol (4 Arm-PEG) coupled histidine (4A-PEG-His) with cobalt metal ion (Co) ²⁺ ) Forming a coordinate bond with the amino group and nitrogen group of histidine, in H ₂ O ₂ Under the action of (C), co ²⁺ From a valence 2 state to a valence 3, i.e. Co ³⁺ At the same time, 4A-PEG-His also changes from liquid to gel-like. Meanwhile, MMP9 is used as a kind of zymolytic protein, which can specifically recognize and hydrolyze special peptide segment, namely 'GALGLP' [34,35 ]]. On the other hand, after myocardial infarction, a series of events results in the pH value of the infarcted area decreasing (pH) due to the continuous death and rupture of cells accompanied by the continuous accumulation of lactic acid<6.8 Is made locally in an acidified environment [36 ]]. The acylhydrazone bond as one of the dynamic covalent bonds is stronger than the intermolecular weak interaction (hydrogen bond, etc.), and is present under a specific pH condition (pH)<6.8 Have reversible properties such that the acylhydrazone bond may be at pH<6.8 environmental fracture [37 ]]. Based on the special property of acylhydrazone bond, a great deal of research has been carried out on utilizing acylhydrazone bond to connect related compounds so that the acylhydrazone bond can be used for connecting the related compounds at pH<6.8 acid environment cleavage, thereby isolating Compound [38]。

The current research is mainly focused on the microenvironment (pH) of the infarcted zone after acute myocardial infarction<6.8 High H in the early stages of infarction), high H in the early stages of infarction ₂ O ₂ Content and later gradual increase of microenvironment MMP9 expression. In the design, the polypeptide which is specifically hydrolyzed and identified by MMP9 is successfully utilized to modify 4A-PEG, and Co is added into the solution ²⁺ After that, do not affect the function of the compound, and are in H ₂ O ₂ Forming a gel-like under the action of (1). Meanwhile, the acyl hydrazone bond is used for connecting DSPE and PEG to form DHPM, an aptamer of CD63 is selected to modify the DHPM, the DHPM is combined with and wraps the gel material, and the gel material is combined with Tregs ^Sp_hi _Exos ^CXCR2_hi Anchoring to form a composite structure. The system successfully targets the infarct area under the action of CXCR2, realizes the rupture of microspheres under acidic condition and utilizes the high H of the infarct area ₂ O ₂ Content of Co in the gel raw material ²⁺ From the divalent state to the trivalent state, thereby forming a gel that immobilizes the exosomes. Then, with a gradual increase in MMP9 in the later stage, the hydrogel is destroyed, causing the exosomes to be gradually released. Achieves the protection effect on acute myocardial infarction as a non-invasive treatment by targeting delivery and fixation and slow release of exosome. The result provides a new choice for treating acute myocardial infarction.

The cited documents are as follows:

1.KawaiK,UchiyamaM,HesterJ,WoodK,IssaF.RegulatoryTcellsfortolerance.HumImmunol.2018May；79(5):294-303.

2.XiaN,LuY,GuM,LiN,LiuM,JiaoJ,ZhuZ,LiJ,LiD,TangT,LvB,NieS,ZhangM,LiaoM,LiaoY,YangX,ChengX.AUniquePopulationofRegulatoryTCellsinHeartPotentiatesCardiacProtectionFromMyocardialInfarction.Circulation.2020Nov17；142(20):1956-1973.

3.QaduraM,TerenziDC,VermaS,Al-OmranM,HessDA.ConciseReview:CellTherapyforCriticalLimbIschemia:AnIntegratedReviewofPreclinicalandClinicalStudies.StemCells.2018Feb；36(2):161-171.

4.WangJ,ZhaoC,XiaoJ.ExosomesinCardiovascularDiseasesandTreatment:ExperimentalandClinicalAspects.JCardiovascTranslRes.2019Feb；12(1):1-2.

5.WuR,GaoW,YaoK,GeJ.RolesofExosomesDerivedFromImmuneCellsinCardiovascularDiseases.FrontImmunol.2019Mar29；10:648.

6.KimMS,HaneyMJ,ZhaoY,YuanD,DeygenI,KlyachkoNL,KabanovAV,BatrakovaEV.Engineeringmacrophage-derivedexosomesfortargetedpaclitaxeldeliverytopulmonarymetastases:invitroandinvivoevaluations.Nanomedicine.2018Jan；14(1):195-204.

7.YangJ,ChenZ,PanD,LiH,ShenJ.UmbilicalCord-DerivedMesenchymalStemCell-DerivedExosomesCombinedPluronicF127HydrogelPromoteChronicDiabeticWoundHealingandCompleteSkinRegeneration.IntJNanomedicine.2020Aug11；15:5911-5926.

8.GarbernJC,MinamiE,StaytonPS,MurryCE.DeliveryofbasicfibroblastgrowthfactorwithapH-responsive,injectablehydrogeltoimproveangiogenesisininfarctedmyocardium.Biomaterials.2011Mar；32(9):2407-16.

9.UthmanL,NederlofR,EerbeekO,BaartscheerA,SchumacherC,BuchholtzN,HollmannMW,CoronelR,WeberNC,ZuurbierCJ.DelayedischaemiccontractureonsetbyempagliflozinassociateswithNHE1inhibitionandisdependentoninsulininisolatedmousehearts.CardiovascRes.2019Aug1；115(10):1533-1545.

10.WagnerMJ,KhanM,MohsinS.HealingtheBrokenHeart；TheImmunomodulatoryEffectsofStemCellTherapy.FrontImmunol.2020Apr9；11:639.

11.SaparovA,OgayV,NurgozhinT,ChenWCW,MansurovN,IssabekovaA,ZhakupovaJ.Roleoftheimmunesystemincardiactissuedamageandrepairfollowingmyocardialinfarction.InflammRes.2017Sep；66(9):739-751.

12.FrangogiannisNG.Cardiacfibrosis:Cellbiologicalmechanisms,molecularpathwaysandtherapeuticopportunities.MolAspectsMed.2019Feb；65:70-99.

13.WuX,ShowiheenSAA,SunAR,CrawfordR,XiaoY,MaoX,PrasadamI.ExosomesExtractionandIdentification.MethodsMolBiol.2019；2054:81-91.

14.PanpanLi,YuguoXia,JingchengHao,andXuWang.TransientHealabilityofMetallosupramolecularPolymerNetworksMediatedbyKineticControlofCompetingChemicalReactions.Macromolecules202053(8),2856-2863

15.LindseyML.Assigningmatrixmetalloproteinaserolesinischaemiccardiacremodelling.NatRevCardiol.2018；15:471-479.

16.KaurJ,MishraV,SinghSK,GulatiM,KapoorB,ChellappanDK,GuptaG,DurejaH,AnandK,DuaK,KhatikGL,GowthamarajanK.Harnessingamphiphilicpolymericmicellesfordiagnosticandtherapeuticapplications:Breakthroughsandbottlenecks.JControlRelease.2021Jun10；334:64-95.

17.Blanco-GómezA,Fernández-

BlancoV,RodríguezJ,PeinadorC,GarcíaMD.ThinkingOutsidethe"BlueBox":InducedFitwithinaUniqueSelf-AssembledPolycationicCyclophane.JAmChemSoc.2019Mar6；141(9):3959-3964.

18.ZhouG,LatchoumaninO,HebbardL,DuanW,LiddleC,GeorgeJ,QiaoL.Aptamersastargetingligandsandtherapeuticmoleculesforovercomingdrugresistanceincancers.AdvDrugDelivRev.2018Sep；134:107-121.

19.RahimizadehK,AlShamailehH,FratiniM,ChakravarthyM,StephenM,ShigdarS,VeeduRN.DevelopmentofCell-SpecificAptamers:RecentAdvancesandInsightintotheSelectionProcedures.Molecules.2017Nov27；22(12):2070.

20.CanouraJ,YuH,AlkhamisO,RoncancioD,FarhanaR,XiaoY.AcceleratingPost-SELEXAptamerEngineeringUsingExonucleaseDigestion.JAmChemSoc.2021Jan20；143(2):805-816.

21.WestmanPC,LipinskiMJ,LugerD,WaksmanR,BonowRO,WuE,EpsteinSE.InflammationasaDriverofAdverseLeftVentricularRemodelingAfterAcuteMyocardialInfarction.JAmCollCardiol.2016May3；67(17):2050-60.

22.EpelmanS,LiuPP,MannDL.Roleofinnateandadaptiveimmunemechanismsincardiacinjuryandrepair.NatRevImmunol.2015Feb；15(2):117-29.

23.XiaN,LuY,GuM,LiN,LiuM,JiaoJ,ZhuZ,LiJ,LiD,TangT,LvB,NieS,ZhangM,LiaoM,LiaoY,YangX,ChengX.AUniquePopulationofRegulatoryTCellsinHeartPotentiatesCardiacProtectionFromMyocardialInfarction.Circulation.2020Nov17；142(20):1956-1973.

24.QaduraM,TerenziDC,VermaS,Al-OmranM,HessDA.ConciseReview:CellTherapyforCriticalLimbIschemia:AnIntegratedReviewofPreclinicalandClinicalStudies.StemCells.2018Feb；36(2):161-171.

25.KalluriR,LeBleuVS.Thebiology,function,andbiomedicalapplicationsofexosomes.Science.2020Feb7；367(6478):eaau6977.

26.ZhangY,LiuY,LiuH,TangWH.Exosomes:biogenesis,biologicfunctionandclinicalpotential.CellBiosci.2019Feb15；9:19.

27.LatifkarA,HurYH,SanchezJC,CerioneRA,AntonyakMA.Newinsightsintoextracellularvesiclebiogenesisandfunction.JCellSci.2019Jul1；132(13):jcs222406.

28.HouZ,QinX,HuY,ZhangX,LiG,WuJ,LiJ,ShaJ,ChenJ,XiaJ,WangL,GaoF.LongtermExercise-DerivedExosomalmiR-342-5p:ANovelExerkineforCardioprotection.CircRes.2019Apr26；124(9):1386-1400.

29.WuR,GaoW,YaoK,GeJ.RolesofExosomesDerivedFromImmuneCellsinCardiovascularDiseases.FrontImmunol.2019Mar29；10:648.

30.KimMS,HaneyMJ,ZhaoY,YuanD,DeygenI,KlyachkoNL,KabanovAV,BatrakovaEV.Engineeringmacrophage-derivedexosomesfortargetedpaclitaxeldeliverytopulmonarymetastases:invitroandinvivoevaluations.Nanomedicine.2018Jan；14(1):195-204.

31.SalunkheS,Dheeraj,BasakM,ChitkaraD,MittalA.Surfacefunctionalizationofexosomesfortarget-specificdeliveryandinvivoimaging&tracking:Strategiesandsignificance.JControlRelease.2020Oct10；326:599-614.

32.WangC,WangM,XuT,ZhangX,LinC,GaoW,XuH,LeiB,MaoC.EngineeringBioactiveSelf-HealingAntibacterialExosomesHydrogelforPromotingChronicDiabeticWoundHealingandCompleteSkinRegeneration.Theranostics.2019Jan1；9(1):65-76.

33.YangJ,ChenZ,PanD,LiH,ShenJ.UmbilicalCord-DerivedMesenchymalStemCell-DerivedExosomesCombinedPluronicF127HydrogelPromoteChronicDiabeticWoundHealingandCompleteSkinRegeneration.IntJNanomedicine.2020Aug11；15:5911-5926.

34.PanpanLi,YuguoXia,JingchengHao,andXuWang.TransientHealabilityofMetallosupramolecularPolymerNetworksMediatedbyKineticControlofCompetingChemicalReactions.Macromolecules202053(8),2856-2863

35.LindseyML.Assigningmatrixmetalloproteinaserolesinischaemiccardiacremodelling.NatRevCardiol.2018；15:471-479.

36.UthmanL,NederlofR,EerbeekO,BaartscheerA,SchumacherC,BuchholtzN,HollmannMW,CoronelR,WeberNC,ZuurbierCJ.DelayedischaemiccontractureonsetbyempagliflozinassociateswithNHE1inhibitionandisdependentoninsulininisolatedmousehearts.CardiovascRes.2019Aug1；115(10):1533-1545.

37.ChenX,GuoL,MaQ,ChenW,FanW,ZhangJ.Design,Synthesis,andBiologicalEvaluationofNovelN-AcylhydrazoneBondLinkedHeterobivalentbeta-CarbolinesasPotentialAnticancerAgents.Molecules.2019Aug14；24(16):2950.

38.LiuY,LiuY,WangQ,HanY,ChenH,TanY.DoublyDynamicHydrogelFormedbyCombiningBoronateEsterandAcylhydrazoneBonds.Polymers(Basel).2020Feb21；12(2):487.

the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A conjugated peptide comprising 4A-PEG modified with a polypeptide comprising histidine and a peptide fragment for enzymatic hydrolysis of MMP9, said peptide fragment for enzymatic hydrolysis of MMP9 having the following SEQ ID NO:2, the metallic cobalt ion is combined with histidine by a coordination bond; the metallic cobalt ion can be based on H ₂ O ₂ In response, the conjugated peptide is gel-like.

2. PH/H ₂ O ₂ A MMP9 time-ordered responsive microsphere, comprising: a microsphere comprising distearoylphosphatidylethanolamine and polyethylene glycol bonded by acylhydrazone bond, wherein the conjugate peptide according to claim 1 is loaded in the microsphere.

3. The pH/H of claim 2 ₂ O ₂ The MMP9 time-sequence response microsphere is characterized in that the acylhydrazone bond is broken under the environment that the pH value is less than 6.8;

preferably, the loading of the microspheres with the conjugated peptide is 32.34 ± 7.43wt%.

4. An exosome-carrying biological vector, characterized in that it comprises the pH/H according to any one of claims 2 to 3 ₂ O ₂ The MMP9 time-sequence response microsphere is connected with a CD63 aptamer at the periphery through an acylhydrazone bond, and the CD63 aptamer is specifically combined with an exosome through a CD63 marker on the surface of the exosome;

preferably, the exosomes are derived from stem cells or regulatory T cells;

preferably, the exosome is derived from adipose mesenchymal stem cell, bone marrow mesenchymal stem cell, umbilical cord mesenchymal stem cell, placenta mesenchymal stem cell, urine-derived stem cell or endothelial progenitor cell;

preferably, the CD63 aptamer specifically binds to exosomes derived from SPARC highly-expressed regulatory T cells through a CD63 marker on the surface of the exosomes;

preferably, the CD63 aptamer specifically binds to exosomes derived from CXCR 2-high-expression and SPARC-high-expression regulatory T cells through a CD63 marker on the surface of the exosomes;

preferably, the regulatory T cells with high CXCR2 expression and high SPARC expression realize the high CXCR2 expression of the regulatory T cells with high SPARC expression by a gene editing means, and finally obtain exosomes with high CXCR2 expression;

preferably, the sequence of the aptamer is as set forth in SEQ ID NO:1 is shown in the specification;

preferably, the aptamer is further modified with a modifier or labeled with a detectable label;

preferably, the modifier is biotin; the detectable label is selected from the group consisting of a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.

5. A pH/H according to any one of claims 2 to 3 ₂ O ₂ The preparation method of the MMP9 time-sequence response microsphere is characterized by comprising the following steps:

mixing the conjugate peptide and microspheres formed by distearoyl phosphatidyl ethanolamine connected by acylhydrazone bonds and polyethylene glycol in a solvent, centrifuging, and vacuum drying;

preferably, the ratio of the concentration of the conjugated peptide in the solvent to the concentration of the microspheres in the solvent is 2.3-2.7mg/ml:4.8-5.2mg/ml;

preferably, the solvent is dichloromethane, and water is added when the conjugated peptide is mixed with the microspheres until the volume of the added water accounts for 30% of the volume of the total mixed solution; preferably, water is added at a rate of 0.5-0.6 mL/h;

preferably, after the water is added to 30% of the volume of the total mixed solution, water accounting for 50% of the volume of the total mixed solution is added at the speed of 2-2.5 mL/h.

6. A method for producing the exosome-carrying biological vector according to claim 4, characterized in that the production method comprises the steps of:

time-ordered response microspheres were mixed with CD63 aptamer at a ratio of 0.23-0.27mM: carrying out mixing reaction at a molar ratio of 0.008-0.012mM to obtain a time-sequence response microsphere connected with a CD63 aptamer through an acylhydrazone bond, and then mixing exosome derived from regulatory T cells with the time-sequence response microsphere connected with the CD63 aptamer;

preferably, before the CD63 aptamer is subjected to mixed reaction, the method further comprises modifying an ethynyl group on the CD63 aptamer and connecting the ethynyl group with the p-azidoacetophenone through point chemistry;

preferably, the mixing mass ratio of the exosome to the time-series response microsphere connected with the CD63 aptamer is 0.5-1.5;

preferably, the mixing is performed at 37 ℃. + -. 0.5 ℃ for 3h of incubation.

7. The conjugated peptide according to claim 1, the pH/H according to any one of claims 2 to 3 ₂ O ₂ Use of MMP9 time-ordered response microspheres or the exosome-loaded biological vector of claim 4 in preparation of a myocardial infarction repair drug.

8. The use according to claim 7, wherein the myocardial infarction is selected from the group consisting of acute myocardial infarction, subacute myocardial infarction, myocardial infarction reperfusion, or a ventricular aneurysm;

preferably, in said use, the pH triggers the release of exosomes, reactive oxygen species (H), of said biological vector ₂ O ₂ ) The oxidation of metallic cobalt ions is initiated to form a gel to immobilize the released exosomes, then the degradation of the gel is triggered based on the hydrolysis of MMP9 enzyme, slowly releasing the exosomes.

9. A medicament for treating myocardial infarction, which comprises the conjugated peptide of claim 1, the pH/H of any one of claims 2 to 3 ₂ O ₂ A/MMP 9 time-ordered response microsphere or exosome-loaded biological vector according to claim 4.

10. The medicament of claim 9, wherein the medicament for treating myocardial infarction further comprises pharmaceutically acceptable additives or auxiliary materials;

preferably, 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.

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