Microneedle patch, preparation method and application thereof in myocardial ischemia-reperfusion injury

Abstract

The application discloses a microneedle patch, a preparation method and application thereof in myocardial ischemia-reperfusion injury, and relates to the technical field of myocardial ischemia. The microneedle patch has clear metal ion-dependent phased release capacity and clear clinical transformation potential: preoperative release of P5CS-containing exosomes enhances ischemia tolerance, and postoperative release of ATP5B-containing exosomes promotes energy metabolism reconstruction. Therefore, the application provides a phased and precise intervention strategy, which can significantly improve the treatment effect and prognosis of myocardial ischemia-reperfusion injury.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of myocardial ischemia, in particular to a microneedle patch, a preparation method and application thereof in myocardial ischemia-reperfusion injury. BACKGROUND

[0002] Myocardial ischemia-reperfusion (MI / R) injury is the main mechanism of secondary injury in the treatment of acute myocardial infarction, and its severity directly affects the prognosis of patients. Mitochondria play a core role in the whole MI / R process, showing biphasic dysfunction: oxidative phosphorylation is impaired during ischemia, and cell metabolism is inhibited; while oxygen reenters during reperfusion, activating the electron transport chain and producing a large amount of reactive oxygen species (ROS). This process leads to the collapse of mitochondrial membrane potential, calcium overload and the opening of the mitochondrial permeability transition pore (mPTP), ultimately inducing programmed cell death. Although current interventions targeting oxidative stress, autophagy activation and mPTP inhibition have shown therapeutic potential in animal experiments, their clinical translation has been limited, mainly due to the lack of stage-specificity and targeted delivery strategies.

[0003] Previous studies have reported that cardiomyocytes exhibit significant heterogeneity in metabolic activity, mitochondrial density and stress response. In the MI / R model, different cardiomyocyte subpopulations exhibit different energy metabolism characteristics. Based on this, previous studies have used spatial single-cell technology to identify specific transcription and metabolic characteristics of surviving cells in the myocardial infarction area, suggesting the existence of a "surviving" subpopulation with natural damage resistance. This finding provides a new approach to identifying and treating cardiomyocytes with anti-damage ability.

[0004] Mitochondria can form functionally heterogeneous subpopulations according to local ATP demand: P5CS-type mitochondria enhance glutamate cycling under oxidative stress to maintain redox homeostasis, while ATP synthase (ATP5B)-type mitochondria are characterized by efficient ATP synthesis. These two mitochondrial subtypes differ significantly in morphology, proteome and functional capacity.

[0005] Although previous studies have attempted to correct metabolic dysfunction through mitochondrial transplantation, the poor stability and low delivery efficiency of free mitochondria limit their application. In contrast, extracellular vesicles (EVs) provide a more feasible system for the stable targeted delivery of mitochondrial components, especially EVs derived from metabolically active cardiomyocytes, which show superior therapeutic potential.

[0006] At the same time, the dynamic changes in local copper and iron ion concentrations during MI / R provide a molecular clue for time-controlled delivery. The concentration of copper ions (Cu 2+ ) increases during ischemia, while the concentration of ferrous ions (Fe 2+) and further exacerbate ROS production.

[0007] Micro-needle patch as a new drug delivery platform, due to its excellent penetration, tissue retention and biocompatibility, has been widely concerned in the field of cardiac targeted therapy in recent years. This kind of device can penetrate the pericardium non-invasively, and directly deliver therapeutic carriers to myocardial tissue, thereby significantly improving the local treatment effect. The micro-needle structure composed of hyaluronic acid (HA), polycaprolactone-polyacrylic acid block copolymer (PCL-b-PAA) and polydopamine (PDA) not only has good functionalization ability, but also has environmental responsiveness, which can be used as a double-metal ion peptide controlled release platform.

[0008] In summary, there is currently a lack of responsive and stage-specific mitochondrial delivery platforms to achieve precise treatment of ischemic heart disease.

[0009] Therefore, the present application is proposed. SUMMARY

[0010] The present application aims to provide a micro-needle patch, a preparation method and its application in myocardial ischemia-reperfusion injury to solve the above technical problems.

[0011] The present application is implemented as follows:

[0012] In a first aspect, the present application provides a micro-needle patch, comprising: a needle body, a base and a barrier layer located on the back of the base, the needle body is located on the base, and the whole composed of the needle body and the base is arranged in an array form on the barrier layer;

[0013] The needle body comprises engineered exosome 1, copper-responsive active peptide and dopamine-modified needle body matrix; the copper-responsive active peptide is covalently cross-linked with quinone group formed by oxidation of dopamine through thiol group to obtain a cross-linked product; and the exosome 1 is embedded in the cross-linked product formed by the copper-responsive active peptide and the dopamine-modified needle body matrix; the engineered exosome 1 contains mitochondria with high expression of P5CS; and the engineered exosome 1 is derived from HEY1 positive myocardial cells with high expression of P5CS; the amino acid sequence of the copper-responsive active peptide is: SH-(repeat unit 1)n-SH, n is 3-5; the repeat unit 1 is selected from a dipeptide or tripeptide containing His;

[0014] The base comprises engineered exosome 2, iron-responsive active peptide and dopamine-modified base matrix, the iron-responsive active peptide is covalently crosslinked with the quinone group formed by oxidation of dopamine through thiol groups to obtain a crosslinked product; and the exosome 2 is embedded in the crosslinked product formed by the iron-responsive active peptide and the dopamine-modified needle body matrix; the engineered exosome 2 contains mitochondria with high expression of ATP5B; and the engineered exosome 2 is derived from HEY1-positive cardiomyocytes with high expression of ATP5B; the amino acid sequence of the iron-responsive active peptide is: SH-(repeat unit 2)n-SH, n is 3-5; the repeat unit 2 is selected from a His-containing HX dipeptide or tripeptide; wherein X is glycine, alanine or serine;

[0015] The barrier layer comprises a hydrogel material.

[0016] In a second aspect, the application further provides a preparation method of the microneedle patch, comprising the following steps: mixing engineered exosome 1, copper-responsive active peptide and dopamine-modified needle body matrix at a mass ratio of (1-5):(3-15):(20-100) to obtain a pre-gel solution; mixing engineered exosome 2, iron-responsive active peptide and dopamine-modified base matrix at a mass ratio of 5-25:1-8:100 to obtain a precursor solution;

[0017] The pre-gel solution is added to a microneedle mold; before solidification, the precursor solution is added; after standing, the hydrogel material for preparing the barrier layer is coated on the surface of the base; after solidification, the mold is removed to obtain the microneedle patch.

[0018] In a third aspect, the application further provides a drug for preventing or treating diseases, comprising the microneedle patch or the microneedle patch prepared by the preparation method; the diseases are selected from myocardial ischemia and / or reperfusion injury, or ischemic heart disease mainly caused by mitochondrial dysfunction / oxidative stress.

[0019] The treatment mode is selected from the group consisting of post-infarction functional recovery and anti-remodeling, PCI, CABG, valve surgery.

[0020] In a fourth aspect, the application further provides the use of the microneedle patch or the microneedle patch prepared by the preparation method in the preparation of a drug for preventing or treating myocardial ischemia and / or reperfusion injury, and the drug is administered before surgery.

[0021] The application has the following beneficial effects:

[0022] This invention provides a microneedle patch employing a "stage-specific" strategy in the treatment of myocardial ischemia and / or reperfusion injury: during the ischemic phase, engineered exosome 1 containing mitochondria with high P5CS expression can be used to enhance stress tolerance; while during the reperfusion phase, engineered exosome 2 containing mitochondria with high ATP5B expression can be used to restore energy metabolism. The inventors discovered that a cardiomyocyte subset with high HEY1 expression (HEY1⁺CMs) is characterized by superior mitochondrial homeostasis; therefore, selecting a cardiomyocyte subset derived from high HEY1 expression exhibits stronger cellular tolerance to ischemic injury. Combining the characteristics of engineered exosome 1, engineered exosome 2, and the HEY1-expressing cardiomyocyte subset, this invention encapsulates these two exosomes within microneedles, the microneedles comprising copper-responsive and iron-responsive peptides. The copper-responsive peptides can be induced by copper ions to dissociate the gel structure of the needle, allowing engineered exosome 1 to be released; particularly during myocardial infarction and reperfusion, copper ions (Cu... 2+ The concentration of copper ions increases rapidly in the early stage of ischemia, inducing copper cell death (cuproptosis). The microneedle patch provided by this invention can achieve early intervention and treatment of ischemia under the induction of copper ions.

[0023] Iron-responsive peptides can be induced by iron ions to promote gel liquefaction, allowing the release of engineered exosomes 2. Following reperfusion, iron ions (Fe...) 2+ / Fe 3+ Increased iron ion concentration triggers ferroptosis. Therefore, the microneedle patch provided by this invention enables post-reperfusion intervention therapy under the induction of iron ions.

[0024] The microneedle patch provided by this invention is highly compatible with the metabolic characteristics of myocardial injury: in the early stage of ischemia, P5CS enrichment in mitochondria helps cardiomyocytes survive by maintaining membrane potential and NADPH generation; during reperfusion, ATP5B enrichment in mitochondria optimizes electron transport chain efficiency and supports ATP synthesis and cell function recovery.

[0025] The microneedle patch provided by this invention can stably adhere to the surface of various organs in a liquid environment. The barrier layer helps the microneedle patch specifically target the heart, helps reduce the diffusion of drugs to non-target tissues, and further improves the accuracy and safety of treatment.

[0026] In summary, the microneedle patch provided by this invention has a clear metal ion-dependent staged release capability and has clear clinical translation potential: preoperative release of Ev (containing P5CS) enhances ischemic tolerance, and postoperative release of Ev (containing ATP5B) promotes energy metabolism reconstruction. This invention provides a staged and precise intervention strategy that can significantly improve treatment efficacy and prognosis. Attached Figure Description

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be considered as a limitation to the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0028] Figure 1 Mechanism diagram of the microneedle patch system provided by the present application for improving mitochondrial function in the Bama miniature pig MI / R model;

[0029] Figure 2 (A) UMAP plot of single-cell RNA sequencing data, showing the annotation of cell types in myocardial tissue from healthy controls and myocardial infarction (MI) patients; (B) Comparison of expression levels of HEY1 in HEY1+ cardiac muscle cells (HEY1_CMs) and regular cardiac muscle cells (CMs), using a violin plot to show that both the control and the MI group are reflected; (C) Pseudo-time trajectory analysis shows the development / differentiation trend of cardiac muscle cells in physiological and pathological states; (D) Confocal microscopy images of iPSC-derived HEY1 high-expression (HEY1+ CMs) and HEY1 low-expression (HEY1- CMs) cardiac muscle cells, recording their synchronous jumping at different differentiation time points; (E-H) Mitochondrial stress test under OGD / R (oxygen-glucose deprivation / reoxygenation) conditions, comparing the metabolic characteristics of HEY1+ CMs and HEY1- CMs, including: (E) ATP production; (F) Basal respiration; (G) Maximum respiratory capacity; (H) Proton leakage; Data are presented as mean ± standard error (SEM); Statistical tests use one-way ANOVA and Tukey post-hoc test: *P<0.05, **P<0.01, ns is no statistically significant difference;

[0030] Figure 3Figure; (A) UMAP visualization of single-cell RNA sequencing (scRNA-seq) data showing the distribution of HEY1 -high-expressing cardiomyocytes (HEY1_CMs) in healthy controls and myocardial ischemia-reperfusion (MI) patients; (B) Number of total cardiomyocytes and HEY1_CMs in control and MI samples; (C) Pseudo-time trajectory analysis of HEY1_CMs in control and MI conditions, revealing their developmental progression; (D-J) HEY1 -high-expressing (HEY1⁺CMs) and -low-expressing (HEY1⁻CMs) cardiomyocytes derived from induced pluripotent stem cells (iPSCs) were exposed to oxygen-glucose deprivation / reoxygenation (OGD / R) treatment (1% FBS, 0.5% O2, 1 hour of hypoxia followed by 24 hours of recovery) to assess mitochondrial function; the assessment included: (D) mitochondrial oxygen consumption rate (OCR), (E) intracellular calcium ion level (Fluo-4 AM fluorescence intensity), (F) mitochondrial membrane potential (MMP), (G) mitochondrial permeability transition pore (mPTP) opening, (H) reactive oxygen species (ROS) production, (I) mitochondrial length, (J) cell death rate; (K) Confocal microscopy imaging using VDAC1 antibody immunostaining to observe mitochondrial morphological changes after HEY1⁺CMs overexpressed P5CS or ATP5B; (L-M) Extracellular vesicles (EVs) were isolated from HEY1⁺CMs under normal conditions [Ev(Nor)], FCCP treatment [Ev(Con)], P5CS overexpression [Ev(MTP5CS)], and ATP5B overexpression [Ev(MTATP5B)]; (L) Nanoparticle tracking analysis (NTA) to detect the particle size distribution of EVs; (M) Transmission electron microscopy (TEM) imaging to show the morphological characteristics of EVs; (N) Western blot analysis of mitochondrial proteins (P5CS, ATP5B, VDAC1) and exosome marker CD9 expression in EV samples; Data are presented as mean ± standard error (SEM); statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test correction; *P<0.05, **P<0.01, ***P<0.005, ****P<0.001; ns indicates no significant difference;

[0031] Figure 4(A) Flow cytometry was used to detect mitochondrial superoxide anion (MitoSOX fluorescence) to compare the oxidative stress levels of HEY1 high and low expression cardiomyocytes under control and OGD / R conditions; (B) Confocal microscopy imaging showed the differences in mitochondrial morphology in HEY1 high / low expression cells under different conditions. HEY1+ cells had a more complete mitochondrial network and longer mitochondrial morphology; (C) Flow cytometry combined with Annexin V / PI double staining was used to analyze cell death and quantify the proportion of HEY1+ and HEY1- cardiomyocyte apoptosis under control and OGD / R conditions;

[0032] Figure 5 Functional mitochondrial exosomes (EVs) enhance energy metabolism, redox homeostasis, and cell survival rate under OGD and OGD / R stress conditions; induced pluripotent stem cell-derived cardiomyocytes were exposed to hypoxia and glucose deprivation (OGD) or hypoxia and glucose deprivation / reoxygenation (OGD / R) conditions, and were treated with conventional exosomes (Ev(Nor)), exosomes containing MTP5CS (Ev(MTP5CS)), or exosomes containing MTATP5B (Ev(MTATP5B)), respectively; multiple parameters were evaluated to detect mitochondrial function and cell viability: (A-B) Mitochondrial respiration (oxygen consumption rate, OCR) was measured by sequentially adding oligomycin (Oligo), FCCP, and rotenone + antimycin A (Rot+Ant); (C-D) Intracellular ATP levels; (E-F) Cytosolic calcium ion concentration was measured using the Fluo-3 AM fluorescent probe; (G-H) Mitochondrial permeability transition pore (mPTP) opening; (I-J) Mitochondrial membrane potential (MMP) was detected by JC-1 fluorescence; (K-L) Mitochondrial superoxide anion production was evaluated by MitoSOX staining; (M-O) Confocal microscopy imaging was used to analyze mitochondrial morphology; (M-N) Quantitative analysis of mitochondrial length; (P-Q) Cell death was detected by Annexin V-propidium iodide staining; data are expressed as mean ± standard error (mean ± SEM); one-way ANOVA and Tukey's post-hoc test were used for statistical comparison; *P<0.05, **P<0.01, ***P<0.005, ****P<0.001;

[0033] Figure 6iPSC-derived cardiomyocytes were subjected to OGD or OGD / R stress, respectively, and treated with different exosomes (Ev(Nor), Ev(MTP5CS), Ev(MTATP5B)); mitochondrial function was evaluated and oxygen consumption rate (OCR) analysis was performed: (A-C) Under OGD conditions, the following OCR parameters were quantified: (A) basal respiration; (B) maximum respiratory capacity; (C) proton leakage; (D-F) corresponding OCR parameters under OGD / R conditions; (G-H) Mitochondrial reactive oxygen species (ROS) levels were analyzed using MitoSOX staining combined with flow cytometry, cell counts for each group are presented in the table on the right; (I) Cell death was evaluated using AnnexinV / PI double staining after different treatments under OGD and OGD / R stress; data are presented as mean ± SEM; statistical analysis was performed using one-way ANOVA + Tukey’s multiple comparison: *P<0.05, **P<0.01, ***P<0.005, ****P<0.001, ns for not significant;

[0034] Figure 7 Results for the construction and performance evaluation of the microneedle (MN) system; (A) Schematic representation of the microneedle patch loaded with two different types of exosomes [Ev(MTP5CS) and Ev(MTATP5B)]; the microneedle system comprises three functional components: the PPC tip (composed of PCL-b-PAA, PDA and copper ion-responsive peptide), the HPF base and matrix (composed of HA, PDA and iron ion-responsive peptide), and the PCA anti-adhesion barrier layer (composed of polyethylene diacrylate, carboxylated cellulose nanocrystals and acrylic acid); (B) Scanning electron microscopy (SEM) images showing the microstructural differences between PPC, HPF and PAC; the PPC structure is relatively compact with visible microporosity; the HPF presents a highly porous and loose network structure; while the PAC shows a dense block-like morphology; scale bar: 5 μm; (C) SEM visualization of the microneedle array highlighting the uniform conical geometry and the aligned individual microneedles; scale bar: left, 500 μm; middle right, 50 μm; (D) Mechanical testing of the microneedles revealed superior piercing performance for PPC, showing a higher load-displacement response; (E) Comparison of the electrical conductivity of HPF and PPC in skin tissue showed no statistical difference, indicating comparable electrical conductivity performance for both; (F) Degradation curves of HPF and PPC under stimulation with different metal ions (Cu 2+ alone or Cu 2+ + Fe 2+ ) showed a clear structural disintegration under ion stimulation; (G) Degradation curves of HPF and PPC under Cu 2+ or Fe 2+(H) Adhesion tests on major organs such as heart, liver, spleen, lung and kidney showed that the microneedle patch could still firmly adhere to the tissue after being soaked in water, without obvious shedding; (I) Cytotoxicity tests on induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) showed that the PPC, HPF and PAC materials did not affect cell viability, proving that the microneedle system had good biocompatibility; (J) After applying microneedles without a PCA isolation layer to the rat heart, in vivo fluorescence imaging was performed by using rhodamine-labeled exosomes, and the results showed that the exosome signal was displayed in both the heart and lung tissues, indicating that there was non-targeted distribution when there was no PCA barrier layer; (K) Double fluorescence tracing of the rat myocardial infarction / reperfusion (MI / R) model heart: PPC loaded with FITC-labeled exosomes, HPF loaded with rhodamine-labeled exosomes, and the back coated with a PCA layer to prevent reverse diffusion; real-time imaging confirmed that the Cu 2+ and Fe 2+ exosomes were released under the stimulation of different ions, and the release behavior was ion-triggered; the data are expressed as mean ± standard error (mean ± SEM); one-way ANOVA and Tukey post-hoc test were used for statistical comparison; ns, no significant;

[0035] Figure 8To enhance the cardiac function and tissue repair capacity of MN (MixEvs) in a myocardial ischemia-reperfusion (MI / R) model in Bama mini-pigs. (A) Schematic diagram of the experimental design: a myocardial ischemia-reperfusion injury model was established in Bama mini-pigs. (B) Intraoperative photos showing the positioning and fixation of the MN (MixEvs) microneedle patch on the surface of the left ventricle. The patch simultaneously carries Ev (MTATP5B) and Ev (MTP5CS) for local delivery to myocardial tissue. The control groups include: free exosome injection group (injection of a 2:1 mixture of Ev (MTATP5B) and Ev (MTP5CS)) and blank microneedle patch group without exosomes. (C) Electrocardiogram (ECG) recording results of animals in each group at 1 day after surgery. (D) The MN (MixEvs) group showed significant reduction in ST segment elevation, indicating recovery of myocardial function. (E) Serum cardiac troponin I (cTnI) levels were detected at 24 hours after surgery, and the MN (MixEvs) group showed significant reduction, indicating reduced myocardial injury. (F-I) At 4 weeks after surgery, cardiac function was evaluated by Doppler and M-mode echocardiography. The MN (MixEvs) group showed significant improvement in the following key cardiac function parameters: (F) E / A ratio (diastolic function indicator); (G) left ventricular ejection fraction (LVEF); (H) left ventricular fractional shortening (LVFS); (I) Representative ultrasound images further confirmed that the diastolic function recovery in the MN (MixEvs) group was superior to that in the control group. (J) Cardiac samples were taken at 4 weeks after surgery for histological fibrosis analysis. (K) The MN (MixEvs) group showed reduced fibrosis area and more orderly myocardial fiber arrangement. (L) Optical mapping results of action potential and calcium transient showed improved electrical activity and calcium handling capacity in the MN (MixEvs) group. (M) In situ TUNEL staining results showed that myocardial cell apoptosis was significantly reduced in the MN (MixEvs) group, indicating its enhanced cell protection effect. Data are presented as mean ± standard error (mean ± SEM). One-way ANOVA and Tukey's post-hoc test were used for comparison between groups. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001; ns indicates no significant difference.

[0036] Figure 9Middle: (A) Serum CK-MB levels were detected at 24 h after treatment, and the MN(MixEvs) group showed a significant decrease, indicating reduced myocardial injury; (B) Echocardiography evaluation showed no significant difference in heart rate (HR) among the groups, indicating that MN(MixEvs) had no adverse effects on heart rate; (C-F) Quantitative analysis of electrophysiological imaging parameters: (C) Action potential activation time; (D) Action potential 90% repolarization time (APD90); (E) Calcium transient activation time; (F) Calcium transient 90% recovery time (CTD90), the MN(MixEvs) group showed significant improvement in function; (G) TUNEL staining showed that the number of apoptotic cells in the infarct myocardial area was significantly reduced after MN(MixEvs) treatment (green: TUNEL positive, blue: DAPI nuclear staining), scale bar: 100 μm; (H-K) Blood biochemical indicators (UA, UREA, γ-GT, AST) were within the normal range, indicating that no systemic toxicity was caused; (L) H&E staining of major organs (heart, liver, spleen, lung, kidney) showed no structural damage or inflammatory cell infiltration, further verifying that the MN(MixEvs) platform has good systemic biocompatibility; scale bar: 100 μm; data are presented as mean ± SEM, statistical analysis as before;

[0037] Figure 10To promote myocardial repair in MN (MixEvs) by regulating mitochondrial function and metabolic pathways; (A) Transmission electron microscopy (TEM) images show ultrastructural changes in mitochondria in myocardial tissue in each treatment group; the mitochondria in the sham operation group (Sham) are complete in structure; the mitochondria in the MI / R group are severely fragmented, and the structure of the cristae is disordered; the mitochondria in the MN (MixEvs) group have good recovery in morphology, and the structure is arranged in an orderly and complete manner; the red square is the enlarged area; the scale bar is 1 μm; (B) Quantitative evaluation of abnormal mitochondrial morphology according to the aspect ratio (aspect ratio) shows that the mitochondrial damage in the MN (MixEvs) group is significantly reduced; (C) Immunoblot analysis of key proteins in mitochondrial biosynthesis and quality control in myocardial tissue: PGC-1a (main regulator of mitochondrial biosynthesis), COX IV (electron transport chain component), PINK1 (mitochondrial damage sensing protein), TOMM20 (outer mitochondrial membrane marker), and other protein expression in the MN (MixEvs) group are improved, indicating the recovery of mitochondrial structure and functional integrity; (D) KEGG pathway enrichment analysis shows that the genes related to oxidative phosphorylation, fatty acid metabolism, tricarboxylic acid (TCA) cycle, HIF-1 signal, and calcium signaling pathway in the MN (MixEvs) group are significantly up-regulated; (E) Gene set enrichment analysis (GSEA) further confirms that MN (MixEvs) treatment can enhance the signal pathways related to oxidative phosphorylation, mitochondrial function, and cellular energy metabolism, revealing the molecular basis of its cardioprotective mechanism; the data are expressed as mean ± standard error (mean ± SEM); statistical analysis was performed using one-way ANOVA and Tukey's post-hoc test; *P<0.05, ****P<0.001; ns indicates no significant difference. DETAILED DESCRIPTION

[0038] Reference will now be made in detail to the embodiments of the application, one or more examples of which are set forth below. Each example is provided as an explanation and not as a limitation of the application. Indeed, it will be apparent to one of ordinary skill in the art that numerous modifications and variations of the present application are possible in light of the above teachings. For example, features described or illustrated as part of one embodiment can be used with another embodiment to yield still a further embodiment.

[0039] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If specific conditions are not indicated in the embodiments, conventional conditions or manufacturer recommended conditions are used. If the used reagents or instruments are not indicated by the manufacturer, they are all conventional products that can be purchased on the market.

[0040] DEFINITIONS

[0041] Microneedle patches are a minimally invasive transdermal drug delivery system, consisting of an array of dozens to hundreds of microneedles in the micrometer scale (usually 50-1500 μm).

[0042] In a first aspect, the present application provides a microneedle patch, comprising: a needle body, a base and a barrier layer on the back of the base, the needle body is on the base, and the whole formed by the needle body and the base is arranged on the barrier layer in an array form;

[0043] The needle body comprises engineered exosome 1, copper-responsive active peptide and dopamine-modified needle body matrix; the copper-responsive active peptide is covalently crosslinked with quinone groups formed by oxidation of dopamine through sulfydryl to obtain a crosslinked product; and the exosome 1 is embedded in the crosslinked product formed by the copper-responsive active peptide and the dopamine-modified needle body matrix; the engineered exosome 1 contains mitochondria with high expression of P5CS; and the engineered exosome 1 is from HEY1-positive cardiomyocytes with high expression of P5CS; the amino acid sequence of the copper-responsive active peptide is: SH- (repeating unit 1) n-SH, n is 3-5; the repeating unit 1 is selected from a His-containing dipeptide or tripeptide for Cu 2+ coordination;

[0044] The base comprises engineered exosome 2, iron-responsive active peptide and dopamine-modified base matrix, the iron-responsive active peptide is covalently crosslinked with quinone groups formed by oxidation of dopamine through sulfydryl to obtain a crosslinked product; and the exosome 2 is embedded in the crosslinked product formed by the iron-responsive active peptide and the dopamine-modified needle body matrix; the engineered exosome 2 contains mitochondria with high expression of ATP5B; and the engineered exosome 2 is from HEY1-positive cardiomyocytes with high expression of ATP5B; the amino acid sequence of the iron-responsive active peptide is: SH- (repeating unit 2) n-SH, n is 3-5; the repeating unit 2 is selected from a HX dipeptide or tripeptide of His; wherein X is glycine, alanine or serine, to maintain Fe 2+ / Fe 3+ coordination ability, X=G / A / S provides flexibility and hydrophilicity; in other embodiments, HK can also be used in small amounts to enhance positive electricity and coordination;

[0045] The barrier layer comprises a hydrogel material.

[0046] The present application provides a microneedle patch, which adopts a "stage-specific" strategy in myocardial ischemia and / or reperfusion injury treatment: during the ischemia period, engineered exosome 1 containing mitochondria with high expression of P5CS can be used to enhance stress tolerance, and the mechanism may be to maintain mitochondrial homeostasis: P5CS-enriched mitochondria resist oxidative stress by maintaining NADP⁺ / NADPH balance; and during the reperfusion period, engineered exosome 2 containing mitochondria with high expression of ATP5B can be used to restore energy metabolism, and the mechanism is that ATP5B-enriched mitochondria promote cell recovery by synthesizing energy.

[0047] The inventors found a subpopulation of cardiomyocytes with high expression of HEY1 (HEY1+ CMs) by single-cell RNA sequencing analysis of myocardial tissue from myocardial infarction (MI) patients and non-MI individuals, which is characterized by having a better mitochondrial homeostasis, and thus the subpopulation of cardiomyocytes with high expression of HEY1 has stronger cell tolerance when facing ischemic injury. In combination with the characteristics of engineered exosome 1, engineered exosome 2 and the subpopulation of cardiomyocytes with high expression of HEY1, the present application encapsulates the above two kinds of exosomes in microneedles, and the composition of the microneedles includes copper-responsive active peptides and iron-responsive active peptides. The copper-responsive active peptides can be induced by copper ions to dissociate the gel structure of the needle body, so that the engineered exosome 1 is released; in particular, during myocardial infarction and reperfusion, the concentration of copper ions (Cu 2+ ) rapidly increases in the early stage of ischemia, inducing cell copper death (cuproptosis), and the microneedle patch provided by the present application can realize early ischemic intervention treatment under the induction of copper ions.

[0048] The iron-responsive active peptides can be induced by iron ions to promote gel liquefaction, so that the engineered exosome 2 is released. After reperfusion, the concentration of iron ions (Fe 2+ / Fe 3+ ) rises, triggering ferroptosis. Therefore, the microneedle patch provided by the present application can realize post-reperfusion intervention treatment under the induction of iron ions.

[0049] The microneedle patch provided by the present application is highly consistent with the metabolic characteristics of myocardial injury: in the early stage of ischemia, P5CS-enriched mitochondria help cardiomyocyte survival by maintaining membrane potential and NADPH generation; in the reperfusion period, ATP5B-enriched mitochondria optimize the efficiency of the electron transport chain, supporting ATP synthesis and cell function recovery.

[0050] The microneedle patch provided by the present application can be stably attached to the surface of various organs in a liquid environment, and the barrier layer helps the microneedle patch specifically target the heart, reducing the diffusion of drugs to non-target tissues, and further improving the accuracy and safety of treatment.

[0051] The repeating unit 1 is selected from GGH, HGH, HGG, GHK, AGH or SGH; and the repeating unit 2 is selected from HG, HH, HS, HA, HGH, GHG or HHG.

[0052] The amino acid sequence of the copper-responsive active peptide is SH-(GGH)n-SH, and n is 3, 4 or 5. The amino acid sequence of the iron-responsive active peptide is SH-(HG)n-SH, and n is 3, 4 or 5.

[0053] In a preferred embodiment of the application, the amino acid sequence of the copper-responsive active peptide is SH-(GGH)n-SH, n is 3, and the amino acid sequence of the iron-responsive active peptide is SH-(HG)n-SH, n is 4.

[0054] The copper-responsive active peptide and the iron-responsive active peptide selected from the above-mentioned number of repetitions (n) have better copper response and iron response effects, respectively.

[0055] Specifically, the copper response is better (GGH, n = 3), the effect is faster, and the burst release is obvious: in the presence of Cu 2+ , the PPC layer rapidly disintegrates and has a higher early release ratio; the Fe 2+ response is not sensitive, and the stage selectivity is good.

[0056] The coordination-mechanical balance is optimal: n = 3 provides a moderate His density and a flexible interval (G), which ensures Cu 2+ triggering through multiple points of coordination, and does not excessively increase the stiffness, which is beneficial for the needle tip to pierce and the gel rheology to form; SH-GGHGGHGGH-SH has been verified to be able to stably crosslink with the PDA quinone group and realize ion-triggered release.

[0057] The iron response is better (HG, n = 4):

[0058] The Fe 2+ response is more sensitive and the timing is matched: the HPF layer significantly degrades and releases exosomes under the Fe 2+ condition; the release lags behind the Cu 2+ layer, which is synchronized with the disease process of the increase in iron ions during the reperfusion period, and realizes “two-stage” delivery.

[0059] The sites are more dense and remain flexible: (HG)n of n = 4 increases the His site density without introducing Cys, enhances the multi-point coordination and network destruction effect on Fe 2+ , and at the same time retains the low modulus and adhesion required by the HA base.

[0060] Therefore, the preferred n = 3 (GGH) and n = 4 (HG) respectively exhibit faster triggering effect, higher initial release ratio, and better stage selectivity under Cu 2+ / Fe 2+ conditions, thereby improving the corresponding treatment effects during the ischemic and reperfusion periods.

[0061] In a preferred embodiment of the application, the part connected to the bottom of the needle body is the upper part of the base, and the part away from the bottom of the needle body is the lower part of the base; wherein the combination of the upper part of the base and the needle body is a truncated cone or a cone;

[0062] The bottom end diameter of the frustum or cone is 100-300 μm; the height of the frustum or cone is 100-500 μm; the array density is 100-1000 needle bodies per square centimeter, and the top end diameter of the frustum or cone is less than 80 μm.

[0063] In a preferred embodiment of the application, the needle body matrix is selected from at least one of polycaprolactone-block-polyacrylic acid, hyaluronic acid, polyvinyl alcohol, polycarboxymethyl cellulose, polyethylene glycol, polylactic acid, polyvinylpyrrolidone, chondroitin sulfate, polylactic acid polyglycolic acid copolymer, silk fibroin, cyclodextrin, zinc hyaluronate and gelatin.

[0064] The base matrix is selected from at least one of sodium hyaluronate-maleimide, hyaluronic acid-hydrazine, hyaluronic acid-aldehyde, hyaluronic acid-catechol, polyethylene glycol-maleimide, polyethylene glycol diacrylate, gelatin methacrylate, gelatin-maleimide, chitosan-aldehyde, chitosan-maleimide, polyvinyl alcohol-catechol, polyvinyl alcohol-aldehyde, polycarboxymethyl cellulose-maleimide, polycarboxymethyl cellulose-aldehyde, alginic acid-aldehyde, alginic acid-hydrazine, alginic acid-catechol, silk fibroin-catechol, collagen-maleimide.

[0065] In a preferred embodiment of the application, the needle body matrix is selected from polycaprolactone-block-polyacrylic acid.

[0066] Experiments show that the hydrogel formed by cross-linking copper-responsive active peptide and dopamine-modified polycaprolactone-block-polyacrylic acid has strong compression resistance and is suitable for penetrating the surface of the myocardium, while the cross-linking product formed by cross-linking iron-responsive active peptide and dopamine-modified sodium hyaluronate-maleimide is soft and deformable, and is more suitable for the beating heart. The electrical conductivity test results show that the electrical conductivity of the two cross-linking products is close to that of normal myocardial tissue.

[0067] In a preferred embodiment of the application, the material of the barrier layer is selected from PCA hydrogel, sodium alginate hydrogel, hyaluronic acid hydrogel, chitosan hydrogel, cellulose hydrogel, collagen hydrogel, polyvinyl alcohol hydrogel, polyethylene glycol hydrogel, poly N-isopropyl acrylamide hydrogel, gelatin / PEG composite hydrogel or chitosan / polyacrylic acid hydrogel.

[0068] In a second aspect, the present application also provides a method for preparing a microneedle patch, comprising the following steps: mixing engineered exosome 1, copper-responsive active peptide and dopamine-modified needle body matrix in a mass ratio of (1-5):(3-15):(20-100) to obtain a pre-gel solution, i.e., according to the total mass ratio of the three mixed pre-gel solution, the engineered exosome 1 accounts for 0.86-17.86 wt.%, the copper-responsive active peptide accounts for 2.78-41.67 wt.%, and the dopamine-modified needle body matrix accounts for 50.00-96.15 wt.%; mixing engineered exosome 2, iron-responsive active peptide and dopamine-modified base matrix in a mass ratio of 5-25:1-8:100 to obtain a precursor solution, i.e., according to the total mass ratio of the three mixed precursor solution, the engineered exosome 2 accounts for 4.42-19.84 wt.%, the iron-responsive active peptide accounts for 0.79-7.08 wt.%, and the dopamine-modified base matrix accounts for 75.19-94.34 wt.%.

[0069] The pre-gel solution is added to the microneedle mold, and before solidification, the precursor solution is added. After standing, the hydrogel material for preparing the barrier layer is coated on the surface of the base. After solidification, the microneedle patch is demolded.

[0070] In an embodiment, the mass ratio of the mixture of engineered exosome 1, copper-responsive active peptide and dopamine-modified needle body matrix is, for example, (1-3):(3-10):(20-80), (1-5):(3-12):(50-100), or (1-5):(5-15):(20-100).

[0071] In an embodiment, the engineered exosome 2, iron-responsive active peptide and dopamine-modified base matrix are mixed in a mass ratio of 5-20:1-6:100, (10-25):(1-8):100, or (5-25):(1-8):100.

[0072] In a preferred embodiment of the application, the barrier layer is cross-linked and solidified by light irradiation, and the hydrogel material for preparing the barrier layer comprises: polyethylene glycol diacrylate, acrylic acid and carboxylated cellulose nanocrystals prepared in a mass ratio of 2-8:1-4:0.3-2.0, and 0.5-2.0 wt% of a photoinitiator; preferably, the ratio of polyethylene glycol diacrylate, acrylic acid and carboxylated cellulose nanocrystals is 4:2:1; and the photoinitiator is 1.0 wt%.

[0073] The light irradiation cross-linking and solidification is irradiated under ultraviolet light for 10-20 minutes. For example, the photopolymerization reaction is carried out by irradiating 365 nm ultraviolet light (10 mW / cm 2 ) for 15 minutes.

[0074] In a preferred embodiment of the application, the engineered exosome 1 and the engineered exosome 2 are prepared by the following method:

[0075] First, cardiomyocytes overexpressing HEY1 are prepared; then, the cardiomyocytes overexpressing HEY1 are transfected with a lentiviral vector carrying P5CS to obtain cardiomyocytes overexpressing P5CS and positive for HEY1; the cardiomyocytes overexpressing HEY1 are transfected with a lentiviral vector carrying ATP5 to obtain cardiomyocytes overexpressing ATP5 and positive for HEY1; the lentiviral vector is provided with FCCP to facilitate the entry of mitochondria into exosomes;

[0076] The cardiomyocytes overexpressing P5CS and positive for HEY1 and the cardiomyocytes overexpressing ATP5 and positive for HEY1 are treated with a mitochondrial uncoupling agent to induce the release of mitochondrial components into exosomes;

[0077] Then, exosomes are extracted to obtain exosome 1 containing mitochondria overexpressing P5CS and exosome 2 containing mitochondria overexpressing ATP5.

[0078] The mitochondrial uncoupling agent is, for example, FCCP, which can induce maximum respiration and significantly induce the release of mitochondrial components into exosomes.

[0079] In a third aspect, the present application also provides a medicament for preventing or treating diseases, which comprises the microneedle patch or the microneedle patch prepared by the preparation method described above.

[0080] The diseases are selected from myocardial ischemia and / or reperfusion injury, or ischemic heart disease mainly caused by mitochondrial dysfunction / oxidative stress.

[0081] The treatment is selected from post-infarction functional recovery and anti-remodeling, PCI, CABG or valve surgery; in particular, the treatment is perioperative myocardial protection for PCI, CABG or valve surgery.

[0082] In an alternative embodiment, the medicament further comprises a pharmaceutically acceptable carrier.

[0083] In one embodiment, the medicament is in the form of a tablet, powder, suspension, gel, granule, nanoparticle, capsule, or powder injection.

[0084] In one embodiment, the above-mentioned medicaments are liquid pharmaceutical formulations, such as solutions, suspensions and gels, which generally contain a liquid carrier, such as water and / or pharmaceutically acceptable organic solvents. In addition, such liquid formulations can also contain pharmaceutically acceptable carriers, such as excipients, diluents, pH adjusting agents, emulsifying or dispersing agents, buffers, preservatives, wetting agents, gelling agents (e.g. methylcellulose), dyes and / or flavoring agents, such as defined above. The medicaments can be isotonic, i.e. they can have the same osmotic pressure as blood. The isotonicity of the medicaments can be adjusted by using sodium chloride and other pharmaceutically acceptable agents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic or organic soluble substances. The viscosity of the liquid composition can be adjusted by pharmaceutically acceptable thickeners, such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer and the like. The preferred concentration of the thickener depends on the selected agent.

[0085] In an alternative embodiment, the above-mentioned medicaments are solid pharmaceutical formulations, such as granular formulations and the like.

[0086] In a fourth aspect, the present application also provides the use of the above-mentioned microneedle patch or the microneedle patch prepared by the above-mentioned preparation method in the preparation of a medicament for preventing or treating myocardial ischemia and / or reperfusion injury, wherein the medicament is administered before surgery.

[0087] The features and characteristics of the present application are further described in detail below in conjunction with the examples.

[0088] We first identified a novel subpopulation of cardiomyocytes marked by high expression of HEY1, which can maintain robust mitochondrial function during MI / R. By inducing the directional differentiation of pluripotent stem cells, we successfully obtained HEY1⁺ cardiomyocytes (HEY1⁺ CMs) and constructed EVs enriched with P5CS or ATP5B mitochondria, respectively. Based on the metal ion response mechanism, we designed a microneedle delivery platform composed of PDA-modified HA or PCL-b-PAA, and developed a highly synchronized release system with the MI / R stage: EVs rich in P5CS were encapsulated in Cu 2+ response area for release during the ischemic period; and EVs rich in ATP5B were encapsulated in Fe 2+ response area for release during the reperfusion period. In the MI / R model of Bama mini-pigs, this system significantly improved mitochondrial function, reduced myocardial necrosis area and improved cardiac function, showing strong clinical transformation potential (see Figure 1 ).

[0089] Example 1

[0090] 1. Materials and methods:

[0091] (1) Single-cell transcriptome analysis

[0092] All bioinformatics analysis was performed using the CeleLensCloud platform provided by Singleron Biotechnologies (https: / / www.celelenscloud.cn). Public human heart single-cell and single-nucleus RNA sequencing (sc / snRNA-seq) datasets were obtained from the Human Cell Atlas Data Coordination Platform (ERP123138), the European Genome-phenome Archive (EGAS00001006374), and the cellxgene portal (https: / / cellxgene.cziscience.com / collections / 8191c283-0816-424b-9b61-c3e1d6258a77). These datasets covered myocardial tissue from individuals with and without myocardial infarction (MI) and could be used for comparative analysis under disease and normal conditions.

[0093] After standard quality control (filtering cells with less than 200 expressed genes or more than 10% mitochondrial transcripts), normalization, and dimensionality reduction, unsupervised clustering was performed using the Louvain algorithm, and visualization was performed using UMAP. Cell type annotation was based on typical marker genes for cardiomyocytes, endothelial cells, fibroblasts, immune cells, and the like. The Wilcoxon rank-sum test was used to evaluate the expression of marker genes in each cluster, and the results were displayed in dot plots and heat maps. By analyzing the distribution of each cell subpopulation in the MI and non-MI groups, the infarction-related changes in cell composition were evaluated. Statistical differences in cell type abundance were analyzed using the chi-square test. To explore the lineage relationships and cell state transitions, Monocle3 was used for pseudotemporal analysis, and cell development trajectories were reconstructed based on highly variable genes. Known biomarkers were used to set the starting state, thereby depicting the differentiation pathways of cell lineages such as cardiomyocytes and fibroblasts.

[0094] (2) Differentiation of hiPSCs into cardiomyocytes (CMs) and HEY1-overexpressing cardiomyocytes (HEY1+ CMs)

[0095] Human induced pluripotent stem cells (hiPSCs) were cultured on Matrigel-coated plates using mTeSR TM1 Medium, maintained under standard conditions (37°C, 5% CO2). Cells were initiated for monolayer differentiation program at about 90% confluency. After initiation of differentiation, cells received RPMI 1640 medium (supplemented with insulin-free B-27) with 6 mM CHIR99021 for 24 hours, followed by 5 mM IWP-2 for 48 hours to modulate Wnt signaling pathway. Next, cells were switched to RPMI / B27 medium with insulin, and medium was changed every 2-3 days. Spontaneously beating cardiomyocytes usually appeared at day 8-10.

[0096] At day 14, metabolic selection was performed using glucose-free, lactate-containing medium for 4 days to enrich cardiomyocytes. The obtained iPSC-derived cardiomyocytes exhibited spontaneous contractile ability and expressed cardiomyocyte-specific markers (e.g., cTnI).

[0097] HEY1 overexpression was achieved by infecting cardiomyocytes with lentiviral vector carrying HEY1 gene (pLV-EF1a-HEY1-IRES-GFP, MOI=10) at day 10, with addition of 8 pg / mL polybrene to improve transfection efficiency. GFP expression was observed 48 hours later. Cells infected with empty vector served as control. HEY1+ CMs were used for subsequent experiments between day 15-20, together with regular CMs.

[0098] (3) Mitochondrial respiratory function was measured using Seahorse XF Analyzer

[0099] To evaluate mitochondrial function under pathological and therapeutic conditions, cells received different treatments before oxygen consumption rate (OCR) measurement. The specific groups were as follows:

[0100] Normal group (control), hypoxia-glucose deprivation treatment (OGD, 4 hours), hypoxia-glucose deprivation-reoxygenation treatment (OGD / R, 4 hours OGD + 12 hours reoxygenation).

[0101] After OGD or OGD / R treatment, cells received treatment with exosomes (EVs) of different origins, including:

[0102] Ev(Nor): exosomes from untreated HEY1+ CMs;

[0103] Ev(MTP5CS): exosomes from FCCP-induced P5CS-high-expressing HEY1+ CMs;

[0104] Ev(MTATP5B): exosomes from FCCP-induced ATP5B-high-expressing HEY1+ CMs.

[0105] Mitochondrial respiration function was determined using Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies) after 12 hours of treatment. Cells were seeded at a density of 20,000 per well in an XF96 plate and allowed to adhere overnight.

[0106] On the day of assay, the original medium was replaced with Seahorse XF assay medium (pH 7.4, containing 10 mM glucose, 2 mM glutamine, 1 mM pyruvate) and incubated at 37°C for 1 hour in a CO2-free environment. The following reagents were then sequentially injected:

[0107] Oligomycin (1 mM): ATP synthase inhibitor;

[0108] FCCP (1 mM): Uncoupler to induce maximum respiration;

[0109] Rotenone (0.5 mM) + Antimycin A (0.5 mM): Inhibitors of electron transport chain complex I and III;

[0110] Based on the measured OCR data, key mitochondrial respiration parameters were calculated, including basal respiration, ATP production, maximum respiratory capacity, and proton leakage.

[0111] (4) Mitochondrial function assessment

[0112] Mitochondrial calcium overload: Cells were incubated with 5 mM Fluo-3 AM (Beyotime) in serum-free medium at 37°C for 30 minutes, then washed with PBS. The fluorescence intensity was then detected by a microplate reader (excitation wavelength: 488 nm; emission wavelength: 525 nm).

[0113] Mitochondrial membrane potential (MMP): Cells were incubated with JC-1 working solution (Beyotime) at 37°C for 20 minutes, then washed with buffer and analyzed in a microplate reader. Red fluorescence (aggregated state) was measured at 525 / 590 nm, green fluorescence (monomeric state) was measured at 490 / 530 nm, and the red / green ratio was used to assess the membrane potential level.

[0114] Mitochondrial permeability transition pore (mPTP) opening: Cells were incubated with 1 mM Calcein-AM and 1 mM CoCl2 (ThermoFisher) for 15 minutes at 37°C, then washed and Calcein fluorescence was detected by a microplate reader (excitation wavelength: 488 nm; emission wavelength: 515 nm). A decrease in fluorescence signal indicates mPTP opening.

[0115] Mitochondrial ROS levels: Cells were incubated with 5 mM MitoSOX™ Red (Thermo Fisher) for 15 min at 37°C in the dark, washed with PBS immediately before detection in a flow cytometer (PE channel).

[0116] Mitochondrial morphology: Cells were incubated with MitoTracker™ Red CMXRos (200 nM, Thermo Fisher) for 30 min at 37°C, fixed in 4% paraformaldehyde, imaged with a confocal microscope, and analyzed for mitochondrial length using ImageJ software (at least 50 cells per group).

[0117] (5) Statistical analysis

[0118] All data are presented as mean ± SD. Comparisons between two groups were made using unpaired two-tailed Student’s t test; comparisons between multiple groups were made using one-way ANOVA followed by Tukey’s post hoc test. Results were considered statistically significant when p value < 0.05. All statistical analyses were performed using GraphPad Prism 9.0 software.

[0119] (6) Conclusion: HEY1+ cardiomyocyte subpopulation maintains mitochondrial homeostasis to enhance resistance to MI / R injury.

[0120] By performing single-cell RNA sequencing analysis on myocardial tissues from myocardial infarction (MI) patients and non-MI individuals, Figure 2 Fig. 1A, we identified a HEY1-high-expressing cardiomyocyte subpopulation (HEY1+ CMs) that was stably present in both groups Figure 3 Fig. 1A, Figure 2 Fig. 1B). Although the total number of cardiomyocytes was significantly reduced after MI, Figure 3 Fig. 1B, suggesting that this subpopulation has stronger tolerance to ischemic injury.

[0121] Pseudo-time analysis showed that cardiomyocytes from non-MI individuals could be divided into three developmental stages, while MI patients only showed two stages Figure 2 Fig. 1C. HEY1+ CMs in both groups were at the earliest stage of development Figure 3 Fig. 1C, exhibiting a primitive phenotype similar to stem cells, which may confer stronger stress adaptation ability and help maintain function after myocardial infarction.

[0122] To verify the functional advantage of HEY1+CMs, we differentiated induced pluripotent stem cells (iPSCs) to obtain HEY1 high expression (HEY1+CM) and low expression (HEY1- CM) cardiomyocytes Figure 2 After 4 hours of low serum and hypoxia and 12 hours of recovery (OGD / R model), we evaluated mitochondrial function. Oxygen consumption rate (OCR) assay showed that HEY1+CMs maintained stable ATP production, basal respiration, maximum respiratory capacity, and proton leakage, while HEY1- CMs exhibited extensive mitochondrial dysfunction, including loss of membrane potential, calcium overload, increased mPTP opening, and excessive ROS production, indicating that HEY1 expression is closely related to mitochondrial homeostasis Figure 3 Figure 3 Figure 2 Figure 2 Figure 4

[0123] Ultrastructural analysis further confirmed that HEY1+CMs had stronger tolerance to OGD / R stimulation. HEY1- CMs exhibited obvious mitochondrial fragmentation and shortening, while HEY1+CMs maintained intact mitochondrial morphology Figure 3 Figure 4 After OGD / R treatment, HEY1- CMs exhibited a large number of cell death, while HEY1+CMs had a lower level of cell death Figure 3 Figure 4

[0124] As a downstream transcription factor of the Notch signaling pathway, HEY1 has been shown to play a key role in regulating stem cell fate, antioxidant stress, and tissue regeneration. Therefore, HEY1+CMs may represent a primitive cardiomyocyte subpopulation with both homeostatic maintenance and stress repair functions.

[0125] In addition, mitochondrial functional heterogeneity was found in HEY1+CMs: some mitochondria were rich in P5CS, involved in glutamine metabolism and redox balance; while others were rich in ATP synthase subunit ATP5B, mainly responsible for ATP synthesis.

[0126] (7) To explore its functional differentiation, this embodiment also constructed P5CS high expression (P5CShigh HEY1+CMs) and ATP5B high expression (ATP5Bhigh HEY1+CMs) cardiomyocytes, and confirmed that mitochondria were rich in target proteins respectively Figure 3

[0127] ​​​​​​​​​Construction of HEY1+ CMs with high expression of P5CS / ATP5B and immunofluorescence detection method are as follows:

[0128] HEY1+ CMs were constructed by lentiviral transduction according to the above method. On this basis, a second round of gene modification was further carried out by lentivirus, and the lentivirus carrying P5CS (pLV-EF1a-P5CS-IRES-mCherry) or ATP5B (pLV-EF1a-ATP5B-IRES-mCherry) gene was infected, the MOI was 10, and 8 μg / mL polybrene was added to improve the transduction efficiency. After 48 hours, mCherry positive cells were observed by fluorescence microscope, and were named as P5CShigh HEY1+ CMs and ATP5Bhigh HEY1+ CMs, respectively. Cells infected with empty vector were used as control group.

[0129] In immunofluorescence detection, cells were seeded on cover glass, fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100, blocked with 5% BSA, incubated at 4°C overnight, and the following primary antibodies were added:

[0130] Anti-P5CS (Abcam);

[0131] Anti-ATP5B (Proteintech);

[0132] Anti-VDAC1 (Proteintech, mitochondrial marker);

[0133] After washing, Alexa Fluor labeled secondary antibody was added and incubated at room temperature for 1 hour. Nuclei were stained with Hoechst. Images were obtained by confocal microscope.

[0134] (8) Extraction and characterization of exosomes (EVs):

[0135] Then EVs were extracted from HEY1+ CMs under different treatment conditions, including: normal culture group Ev(Nor), FCCP stimulation group Ev(Con), P5CShigh group Ev(MTP5CS), ATP5Bhigh group Ev(MTATP5B).

[0136] The specific extraction and characterization method of exosomes (EVs) is as follows:

[0137] Exosomes derived from cardiomyocytes were collected after 48 hours of culture in exosome-depleted fetal bovine serum (Gibco) containing medium. The specific treatment is as follows:

[0138] Ev(Nor): from untreated HEY1+ CMs;

[0139] Ev(Con): HEY1⁺CMs were obtained after FCCP treatment (2μM, 4 hours);

[0140] Ev(MTP5CS): P5CShigh HEY1⁺CMs processed by FCCP;

[0141] Ev(MTATP5B): ATP5Bhigh HEY1⁺CMs treated with FCCP.

[0142] After collecting the conditioned medium, the cells and debris were removed by centrifugation (300×g, 10 min; 2000×g, 20 min) sequentially, filtered (0.22 μm), and then ultracentrifuged (100,000×g, 70 min, 4°C). The precipitate was washed with PBS and ultracentrifuged again. The final exosomes were resuspended in PBS and stored at -80°C.

[0143] Exosome particle size and concentration were determined by nanoparticle tracking analysis (NTA). Morphological observation was performed using transmission electron microscopy (TEM): samples were dropped onto a copper mesh, negatively stained with 2% uranium acetate, and then air-dried before TEM imaging.

[0144] Protein content was quantified using the BCA (Thermo Fisher) method. Equal volumes of protein were loaded for SDS-PAGE electrophoresis, transferred to a PVDF membrane, and detected by Western blotting using the following primary antibodies:

[0145] Anti-P5CS (Proteintech): Assessment of mitochondrial P5CS enrichment;

[0146] Anti-ATP5B (Proteintech): Detects ATP synthase subunits;

[0147] Anti-VDAC1 (Proteintech): a marker of total mitochondrial load;

[0148] Anti-CD9 (Proteintech): A surface marker of EVs, used as an internal reference.

[0149] Incubation with HRP-labeled secondary antibody followed by ECL substrate development and detection.

[0150] FCCP, as a mitochondrial uncoupling agent, significantly induces the release of mitochondrial components into EVs, leading to an increase in EV particle size. Figure 3 L in the middle, Figure 3 M). Western blot results further confirmed that Ev(MTP5CS) and Ev(MTATP5B) are rich in the target mitochondrial protein (MTP5CS). Figure 3 The N diagram in the figure shows that we have successfully isolated a functionally specific mitochondrial EV subtype.

[0151] These results indicate that HEY1⁺CMs possess a survival advantage in ischemia-reperfusion environments, and the mechanism may lie in maintaining mitochondrial homeostasis: P5CS-enriched mitochondria resist oxidative stress by maintaining the NADP⁺ / NADPH balance, while ATP5B-enriched mitochondria promote cellular recovery through energy synthesis. Furthermore, EVs, as stable mitochondrial carriers, may protect ischemic myocardium through paracrine mechanisms; this concept has been preliminarily confirmed in recent research on mitochondrial transfer and regenerative medicine.

[0152] In summary, this invention reveals the developmental characteristics, functional advantages, and anti-damage mechanisms of the HEY1⁺CM subpopulation, and proposes an innovative cardioprotective strategy based on subcellular structure and secretory products.

[0153] (9) Exosomes encapsulating heterogeneous mitochondria exhibit differentiated cardioprotective effects at different stages:

[0154] To investigate the functional differences of HEY1⁺CMs-derived exosomes (EVs) rich in P5CS or ATP5B mitochondria during ischemia and reperfusion, we established hypoxia / glucose deprivation (OGD) and OGD / R models in iPSC-derived cardiomyocytes for validation.

[0155] In the OGD model, exosomes Ev (MTP5CS) rich in P5CS mitochondria significantly improved mitochondrial respiratory function in cardiomyocytes, including indicators such as ATP production, basal respiration, maximum respiratory capacity, and proton leakage. Figure 5 China A Figure 5 C in the middle Figure 6 China A- Figure 6 Ev(MTP5CS) can also effectively alleviate mitochondrial stress responses under ischemic conditions, such as Ca2+. 2+ Overload, mPTP opening, membrane potential loss, and ROS generation ( Figure 5 E, Figure 5 China G, Figure 5 Middle I, Figure 5 Zhong K, Figure 6 In addition, this exosome also inhibited OGD-induced mitochondrial fragmentation and reduced cardiomyocyte death (G). Figure 5 M, Figure 5 O, Figure 5 (China P).

[0156] In contrast, the protective effect of mitochondrial exosomes rich in ATP5B, Ev(MTATP5B), was relatively limited during the OGD phase. However, in the OGD / R model, Ev(MTATP5B) exhibited a significant protective effect. It not only improved mitochondrial respiratory function in cardiomyocytes (… Figure 5 B,Figure 5 Mid-D, Figure 6 Mid-D- Figure 6 Mid-F), also alleviated Ca 2+ overloading, inhibited mPTP opening, restored membrane potential, and reduced ROS levels ( Figure 5 Mid-F, Figure 5 Mid-H, Figure 5 Mid-J, Figure 5 Mid-L, Figure 6 Mid-H). The exosome also effectively prevented OGD / R-induced mitochondrial fragmentation, thereby protecting cardiomyocytes and reducing cell death ( Figure 5 Mid-N, Figure 5 Mid-O, Figure 5 Mid-Q). Conversely, Ev(MTP5CS) failed to provide similar protective effects during the reperfusion phase.

[0157] This phase-dependent difference in protection may be due to the difference in metabolic demand of cardiomyocytes during ischemia and reperfusion. During the ischemic phase, mitochondrial electron transport chain (ETC) activity decreases, ATP synthase cannot function normally, and ATP5B-enriched mitochondria cannot effectively produce energy, which may exacerbate oxidative stress. However, P5CS-enriched mitochondria can alleviate oxidative damage by maintaining membrane potential and NADPH synthesis, supporting the survival of cardiomyocytes under hypoxic conditions9]. During the reperfusion phase, oxygen and nutrients are rapidly restored, and oxidative phosphorylation (OXPHOS) becomes the main source of ATP, at which time ATP5B-enriched mitochondria can significantly improve ETC efficiency, rapidly restore cellular energy metabolism and homeostasis.

[0158] This finding has important clinical implications, suggesting that a phased and precise intervention strategy can be implemented in the treatment of acute myocardial infarction: Ev(MTP5CS) can be given before percutaneous coronary intervention (PCI) to enhance the tolerance of cardiomyocytes to ischemia, and Ev(MTATP5B) can be given after PCI to promote energy metabolism recovery and cardiac function reconstruction, thereby optimizing treatment outcomes and prognosis.

[0159] Example 2

[0160] This example prepares the matrix material of the needle body, the needle body, the matrix material of the base, and the base.

[0161] (1) Synthesis of PPC and PPC_Ev(MTP5CS)

[0162] The 10 mmol of ε-caprolactone (ε-CL, purity 99%) was dissolved in 50 mL of anhydrous toluene, 0.1 mmol of 2-mercaptoethanol was added as an initiator, and 0.05 mmol of benzoyl peroxide (BPO) was added as a free radical initiator. The reaction was carried out at 120°C for 12 hours under nitrogen protection. After the reaction was completed, the solution was cooled to room temperature and poured into an excess of cold ethanol to precipitate, and the resulting product was centrifuged after being washed with ethanol several times to obtain a thiol-terminated poly-caprolactone (HS-PCL).

[0163] Subsequently, 1 gram of HS-PCL was dissolved in 20 mL of dry N,N-dimethylformamide (DMF), 10 mmol of acrylic acid (AA, purity 99%) and 0.1 mmol of azobisisobutyronitrile (AIBN) were added as a thermal initiator. The reaction was carried out at 70°C for 6 hours under nitrogen protection. The product was precipitated into a large amount of cold diethyl ether, washed and dried under vacuum to obtain block copolymer PCL-b-PAA. The synthesis was confirmed by hydrogen spectrum (^1H NMR, solvent CDCl3) to be successful.

[0164] To graft dopamine, PCL-b-PAA was dissolved in MES buffer (pH 5.5), EDC and NHS (molar ratio 1:1) were added to activate the carboxyl group on PAA, and the reaction was carried out at room temperature for 30 minutes. Another dopamine hydrochloride was dissolved in PBS (pH 7.2) and slowly added to the activated PCL-b-PAA solution, and stirred gently in the dark for 6 hours to form stable amide bonds. To avoid oxidation of dopamine, 0.1 mM ascorbic acid was added to the reaction. The product after the reaction was dialyzed to remove unreacted substances and by-products, and dopamine-functionalized PCL-b-PAA was obtained.

[0165] To form a dynamic cross-linked structure, 200 μM of SH-GGHGGHGGH-SH peptide was dissolved in PBS (pH 7.4) and co-incubated with dopamine-modified PCL-b-PAA hydrogel precursor (3 mg / mL) for 6 hours. In this process, the thiol group of the peptide and the quinone group formed by the oxidation of dopamine were covalently cross-linked by Michael addition reaction to form a PPC hydrogel. Multiple PBS washes were performed to remove unbound peptides.

[0166] For the preparation of exosome-loaded PPC (PPC_Ev(MTP5CS)), the exosomes Ev(MTP5CS) enriched with P5CS mitochondria were suspended in PBS and mixed with SH-GGHGGHGGH-SH peptide and dopamine-modified PCL-b-PAA precursor. The mass ratio of Ev(MTP5CS), SH-GGHGGHGGH-SH peptide and dopamine-modified PCL-b-PAA precursor was 3:9:60, and the mass fraction of Ev(MTP5CS), SH-GGHGGHGGH-SH peptide and dopamine-modified PCL-b-PAA precursor was 4.17 wt.%, 12.50 wt.% and 83.33 wt.% respectively, based on the total mass of the mixture. During the in-situ gelation process, the exosomes were embedded in the peptide-dopamine cross-linked network formed. The final hydrogel was slightly dried at low temperature or directly kept in a hydrated state to obtain PPC_Ev(MTP5CS) that can be used for the tip part of the microneedle, i.e. the needle body.

[0167] (2) Synthesis of HPF and HPF_Ev(MTATP5B)

[0168] Sodium hyaluronate (HA) with a molecular weight of about 100 kDa was dissolved in MES buffer (pH 5.5, concentration of 2 mg / mL) and continuously stirred to fully dissolve. Then EDC and NHS were added in turn (molar ratio of 1.2:1) to activate the carboxyl groups on the HA backbone. After 15 minutes, N-(2-aminoethyl) maleimide (AEM) was added, and the molar ratio of AEM to carboxyl groups on HA was 1:1. After 4 hours of reaction at room temperature, the amino group on AEM formed a stable amide bond with the NHS-activated carboxyl group on HA, and maleimide-functionalized HA (HA-Mal) was obtained. The reaction solution was dialyzed (MWCO 10 kDa) to remove unreacted substances and by-products, and freeze-dried to obtain a light yellow HA-Mal powder.

[0169] To introduce the catechol functional group, dopamine hydrochloride (dopamine·HCl) was dissolved in PBS (pH 7.4, final concentration 2 mg / mL) and slowly added to a 1 mg / mL HA-Mal solution. The reaction was carried out at room temperature for 6 hours in the dark. The catechol group in the dopamine molecule was converted to a quinone group during the autoxidation process, and simultaneously covalently combined with the maleimide group on HA-Mal through Michael addition reaction to form dopamine-grafted HA (PDA-HA). The resulting product was stored in the refrigerator for later use.

[0170] To form the hydrogel structure, SH-HGHGHGHG-SH peptide (200 mM) was dissolved in PBS (pH 7.4) and added to PDA-HA solution at a concentration of 3 mg / mL. Incubate at room temperature for 6 hours, the two ends of the peptide thiol group and PDA quinone group form covalent thioether cross-linking through Michael addition, and a dynamic responsive hydrogel structure is constructed, named HPF. Multiple PBS washes to remove unbound peptides.

[0171] To prepare HPF_Ev(MTATP5B), ATP5B-rich mitochondrial exosomes Ev(MTATP5B) were resuspended in PBS and incubated with SH-HGHGHGHG-SH peptide and PDA-HA precursor solution at a mass ratio (based on base matrix = 100): matrix (PDA-HA precursor solution): iron-responsive peptide (SH-HGHGHGHG-SH peptide): exosome protein (Ev(MTATP5B)) = 100:3:12. Based on the total mass of the mixture, the base matrix is 86.96 wt.%, the iron-responsive peptide is 2.61 wt.%, and the exosome protein is 10.43 wt.%. Cross-linking in situ under gentle stirring. During this process, the exosomes are physically embedded and fixed in the hydrogel network. The resulting composite hydrogel can be lightly dried at low temperature or used directly in a hydrated state, constituting the base and main body area of the microneedle patch, i.e. HPF_Ev(MTATP5B), which is the base.

[0172] (3) Synthesis of PCA hydrogel barrier layer

[0173] Cellulose nanocrystals (CNC) were dispersed in deionized water at a mass fraction of 1% (total volume 200 mL) and uniformly dispersed by ultrasonic treatment for 30 minutes. Then, the following oxidation components were added in sequence under continuous stirring to achieve carboxyl modification:

[0174] 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO, 0.1 mmol / g CNC), sodium bromide (NaBr, 1 mmol / g CNC), sodium hypochlorite (NaClO, 5 mmol / g CNC, added dropwise).

[0175] During the reaction, the pH of the system was maintained at 10.0 with 0.5 M NaOH, and the reaction was carried out at room temperature for 4 hours. After the reaction was completed, ethanol was added to terminate the reaction. The resulting mixture was centrifuged at 12,000 rpm for 10 minutes, the precipitate was washed repeatedly with deionized water, and dialyzed in a MWCO 10 kDa dialysis bag in ultrapure water for 48 hours to remove residual salt and reagent, obtaining a carboxylated cellulose nanocrystal (C-CNC) suspension for subsequent use.

[0176] To prepare the hydrogel precursor solution, polyethylene glycol diacrylate (PEGDA, Mn ~ 700 Da), acrylic acid (AA) and C-CNC suspension were mixed at a mass ratio of 4:2:1. PEGDA served as the main cross-linked polymer network, AA provided hydrophilicity and reactive carboxyl groups, and C-CNC acted as a nanoscale rigid skeleton while enhancing the barrier density.

[0177] To the mixed solution, photoinitiator Irgacure 2959 (2% mass fraction) was added and stirred in the dark for 30 minutes to fully dissolve and mix. The prepolymer solution was slowly poured into a pre-cleaned PDMS mold and subjected to photopolymerization under 365 nm ultraviolet light (10 mW / cm 2 ) irradiation for 15 minutes to form a PCA hydrogel film. After demolding at room temperature, the resulting hydrogel film was rinsed thoroughly with sterile PBS to remove unreacted monomers and residual photoinitiator. The resulting hydrogel film was the PCA.

[0178] (4) Preparation of microneedle patches (MN and MN(MixEvs))

[0179] To prepare the microneedle patch, first the PPC pre-gel solution or PPC mixture loaded with exosomes (PPC_Ev(MTP5CS)) was dispersed in the aqueous phase to form a flow state. A precision micropipette was used to slowly inject 20 μL of the mixture into each microneedle hole of a polydimethylsiloxane (PDMS) mold. The mold contained a 10x10 array of microneedle structures, each with a height of about 300 μm and a base diameter of about 150 μm. After loading, the mold was immediately centrifuged at 3000 rpm for 5 minutes at 4°C to ensure that the tip area was fully filled.

[0180] Before the PPC layer had solidified, a second layer of HPF precursor solution (which may or may not contain Ev(MTATP5B)) was quickly added on top of it, about 60 μL per hole to fully fill the lower area of the needle body. Subsequently, centrifugation was performed again at 2000 rpm for 3 minutes to promote the fusion and penetration of the interface between the PPC and HPF layers.

[0181] The mold was then transferred to a 4°C environment or ice bath for 4 hours to partially cross-link and stabilize the double-layer structure. A PCA prepolymer solution was prepared separately by mixing polyethylene glycol diacrylate (PEGDA), acrylic acid (AA), and carboxylated cellulose nanocrystals (C-CNCs) at a mass ratio of 4:2:1, supplemented with 1 wt% photoinitiator Irgacure 2959. While the HPF layer was still in a hydrated state, the PCA solution was gently applied to the back of the mold to form a uniform coating, and a sterile glass sheet was used to gently press the coating to ensure a smooth contact surface.

[0182] Subsequently, ultraviolet light (365 nm, 10 mW / cm2 Irradiate for 10 minutes to photocrosslink and cure the PCA barrier layer. After crosslinking, stabilize the entire three-layer structure at 4°C for 1 hour to ensure that each layer of the network is fully formed.

[0183] Finally, the PDMS mold was slowly peeled off to obtain complete microneedle patches (denoted as MN or MN(MixEvs) depending on whether they contain exosomes). The final product was freeze-dried at -40°C for 12 hours for long-term storage or further experimental applications.

[0184] (5) Characterization of microneedle materials and structures

[0185] The compressive strength of PPC and HPF hydrogels was evaluated using a universal testing instrument (Instron 3343, USA). Cylindrical samples with a diameter of 6 mm and a height of 6 mm were prepared from the hydrogels and compressed at a rate of 1 mm / min at room temperature. The compressive modulus was calculated based on the linear range of the stress-strain curve.

[0186] The conductivity of the hydrogels was determined using a four-probe testing system (Keithley 2400, Lucas Labs S-302-4). PPC and HPF hydrogels were fabricated into disks with a diameter of 10 mm and a height of 2 mm. After pre-equilibration in PBS, conductivity was measured at room temperature and calculated using a standard geometric correction factor.

[0187] PPC, HPF, and PCA hydrogels were all freeze-dried, fractured, and sputter-coated with gold. Imaging was performed using a field emission scanning electron microscope (FE-SEM, Hitachi SU8010, 5kV) to observe their cross-sectional morphology, pore distribution, and network structure. Simultaneously, the microneedle array was imaged to evaluate its tip geometry, surface integrity, and interfaces between layers.

[0188] Freshly excised rat heart, liver, spleen, lung, and kidney tissues were rinsed with PBS and placed on moistened filter paper. The microneedle patch was gently pressed onto the surface of each organ for 30 seconds, then the tissue and patch were immersed together in PBS and gently shaken for 5 minutes. The adhesion ability (retention or detachment) was determined by visually inspecting whether the microneedles remained attached to the tissue surface.

[0189] (6) Metal ion responsive microneedle system realizes phased controlled release of exosomes encapsulated by functional heterogeneous mitochondria.

[0190] a. Stability assessment of ion-responsive gels of PPC and HPF

[0191] PPC and HPF hydrogel samples without exosomes were prepared. Each 500 μL uncrosslinked precursor solution was added into a sterile glass vial (10 mL volume) and crosslinking was completed under standard conditions to form stable hydrogels.

[0192] After gel formation, each vial was treated as follows:

[0193] 1 mL 100 μM CuCl2solution (PBS, pH 7.4) was added;

[0194] 1 mL 100 μM CuCl2+ 100 μM FeSO4mixed solution was added;

[0195] 1 mL PBS (as a control only) was added;

[0196] All samples were incubated at 37 °C for 2 hours. After incubation, the vials were gently inverted for 10 seconds and the gel appearance changes, including gel deformation, liquefaction, and whether it flowed along the vial wall, were recorded by photography.

[0197] If obvious flow or dripping was observed, it was considered as a sign of partial or complete gel destabilization.

[0198] b. Cu 2+ with Fe 2+ Exosome release experiment under stimulation

[0199] HEY1+CMs-derived exosomes were labeled with fluorescein isothiocyanate (FITC) and Rhodamine B, respectively, and encapsulated into PPC and HPF hydrogels, respectively. FITC-labeled exosomes were used for PPC, and Rhodamine B-labeled exosomes were used for HPF. After labeling, the samples were purified by ultracentrifugation and resuspended in PBS, and the final exosome concentration in the precursor solution was adjusted to about 50 μg / mL.

[0200] Each 100 μL of hydrogel precursor solution was added to a 96-well plate, and crosslinking was completed to form stable hydrogels. Incubation was performed at 37 °C with slight shaking. At different time points, the supernatant was collected and subjected to the following conditions, respectively:

[0201] No stimulation (PBS control);

[0202] CuCl2was added;

[0203] FeSO4was added;

[0204] The fluorescence intensity in the supernatant was detected by a microplate reader:

[0205] FITC: excitation / emission wavelength was 488 / 520 nm;

[0206] Rhodamine B: excitation / emission wavelength at 552 / 580 nm;

[0207] The fluorescence reading was converted to the released exosome concentration according to the established standard curve.

[0208] In addition, the release and diffusion process was qualitatively observed and visualized by fluorescence microscopy.

[0209] c. Biocompatibility evaluation

[0210] iPSC-derived cardiomyocytes (iPSC-CMs) were cultured under standard culture conditions (37°C, 5% CO2) and seeded in 24-well plates at a density of 2x10 5 cells per well. Each well was placed with a sterile hydrogel disc, 8 mm in diameter and 1 mm in thickness, pre-equilibrated with culture medium overnight. The hydrogels used included PPC, HPF, and PCA. The wells without hydrogel placement served as blank control group.

[0211] The cells were co-cultured with hydrogels for 168 hours. At time points of 0, 24, 72, 120, and 168 hours, CCK-8 reagent was added to each well at a volume of 10% of the culture medium. After 2 hours of incubation, the absorbance value (450 nm) was measured to assess cell activity.

[0212] d. In vivo fluorescence imaging of microneedle behavior

[0213] After establishing the rat myocardial infarction model following left anterior descending branch (LAD) ligation, PPC and HPF double-layer microneedle patches containing Rhodamine B (without PCA) were applied to the infarct area. Rats were sacrificed at 0, 1, 3, 5, 7, and 15 days, and major organs such as heart, liver, spleen, lung, and kidney were taken. In vitro fluorescence imaging was performed by IVIS imaging system (excitation / emission wavelength at 540 / 580 nm) to observe the distribution of Rhodamine in vivo.

[0214] In another experiment, the microneedle patch contained a FITC-labeled PPC layer and a Rhodamine-labeled HPF layer, covered with a PCA barrier layer. After applying the patch on the rat myocardial infarction model, organs (heart, liver, spleen, lung, kidney) were taken at 0, 1, 3, 5, 7, 9, and 15 days for IVIS imaging (FITC: 488 / 520 nm; Rhodamine: 540 / 580 nm). The results showed:

[0215] FITC signal decayed early, reflecting the responsive degradation of the PPC layer to Cu 2+ ;

[0216] Rhodamine signal decayed delayed, indicating the HPF layer's response to Fe 2+The responsive degradation process of the hydrogel.

[0217] Experimental results:

[0218] During myocardial infarction and reperfusion, copper ion (Cu 2+ ) concentration rapidly increases in the early stage of ischemia, inducing cell copper death (cuproptosis); while after reperfusion, iron ion (Fe 2+ / Fe 3+ ) concentration rises, triggering ferroptosis. Based on this, the above designs an intelligent microneedle patch system responsive to metal ions, which can achieve two-stage exosome delivery in pathological environment.

[0219] The copper-responsive peptide "GGHGGHGGH" is selected and cross-linked with PDA-modified PCL-b-PAA to form PPC hydrogel for loading Ev(MTP5CS) and positioning at the microneedle tip; while the iron-responsive peptide "HGHGHGHG" is cross-linked with PDA-modified HA to form HPF hydrogel for encapsulating Ev(MTATP5B) to constitute the needle body bottom and the base layer. To prevent exosomes from spreading to non-target tissues, especially the lungs, we add a high-density PCA (composed of polyethylene glycol diacrylate, carboxylated cellulose nanocrystals and acrylic acid) barrier layer on the back of the microneedle ( Figure 7 A).

[0220] Structural characterization shows that the PPC hydrogel has good mechanical strength, suitable for use in the microneedle tip, and has enough pores for Ev(MTP5CS) loading; the HPF hydrogel is more flexible, suitable for the myocardial beating environment, and can effectively carry Ev(MTATP5B); the PCA layer has excellent shielding property ( Figure 7 B). Scanning electron microscopy results show that the microneedle structure is uniform and conical, with a needle length of about 300 μm and a bottom diameter of about 150 μm, meeting the requirements of transdermal and myocardial implantation ( Figure 7 C). Mechanical property tests show that PPC has strong compressive resistance, suitable for penetrating the myocardial surface, while HPF is soft and deformable, more suitable for heart beating ( Figure 7 D). Electrical conductivity test results show that the electrical conductivity of PPC and HPF is close to that of normal myocardial tissue ( Figure 7 E).

[0221] Response release experiments show that copper ions can induce the dissociation of the initial gel structure, and iron ions further promote the gel liquefaction ( Figure 7 F). Fluorescence release experiments further confirm that the system has clear metal ion-dependent phased release capability ( Figure 7 G). In addition, the microneedle patch can be stably attached to the surface of various organs, including heart, liver, spleen, lung and kidney (Figure 7 H).

[0222] Biocompatibility test showed that PPC, HPF and PCA had no significant change in cell activity after co-cultured with iPSC-derived cardiomyocytes Figure 7 I). In vivo fluorescence imaging verified the barrier effect of PCA: without PCA, the exosome signal was distributed in the heart and lung, while with PCA barrier, the signal was limited to the heart area Figure 7 J, Figure 7 K). Further double fluorescence tracking of FITC-labeled PPC and rhodamine-labeled HPF on the heart of MI / R rats showed that the former was released rapidly in the early stage and the signal decayed rapidly, while the latter was significantly released in the later stage, which clearly supported the "two-stage release" strategy based on metal ion gradient Figure 7 K).

[0223] This system is highly consistent with the metabolic characteristics of myocardial injury: in the early stage of ischemia, P5CS-rich mitochondria help cardiomyocytes survive by maintaining membrane potential and NADPH generation; in the reperfusion period, ATP5B-rich mitochondria optimize the efficiency of the electron transport chain, supporting ATP synthesis and cell function recovery.

[0224] This system has clear clinical translation potential: Ev(MTP5CS) is released before PCI to enhance ischemic tolerance, and Ev(MTATP5B) is released after PCI to promote energy metabolism reconstruction, constructing a phased and precise intervention strategy that significantly improves treatment effect and prognosis.

[0225] Example 3

[0226] Myocardial ischemia-reperfusion model and therapeutic intervention.

[0227] (1) The experimental method of myocardial ischemia-reperfusion model and therapeutic intervention is as follows:

[0228] Bama miniature pigs weighing 15-20 kg were selected, provided by Chengdu Duosi Experimental Animal Co., Ltd. All operations were approved by the Animal Ethics Committee of Sichuan Provincial People's Hospital. Fasting for 12 hours before operation.

[0229] The anesthesia method is as follows:

[0230] Ketamine (10 mg / kg) and xylazine (2 mg / kg) were injected intramuscularly to induce anesthesia;

[0231] After tracheal intubation, connect the respirator;

[0232] General anesthesia maintenance: inhale 1.5-2% isoflurane and oxygen.

[0233] A left thoracotomy was performed at the fourth intercostal space, and the heart was exposed. After opening the pericardium, the left anterior descending (LAD) coronary artery was located 1-1.5 cm below the left atrial appendage.

[0234] Ischemia-reperfusion (I / R) model construction:

[0235] The LAD was encircled and ligated with 4-0 silk thread, and ischemia was confirmed by local cyanosis and ST segment elevation on electrocardiogram.

[0236] After 60 minutes of ischemia, the ligature was loosened, and reperfusion was initiated.

[0237] Animals were randomly divided into five groups:

[0238] Sham group: pass through under the LAD but not ligate, only simulate the operation stress with a sterile needle touching the surface of the heart;

[0239] Saline group: I / R + 200 μL of normal saline was added at the infarct site;

[0240] MN group: I / R + implantation of blank microneedle patches;

[0241] MixEvs group: I / R + direct addition of mixed exosome solution (Ev(MTP5CS):Ev(MTATP5B) = 1:2);

[0242] MN(MixEvs) group: I / R + implantation of microneedle patches loaded with mixed exosomes.

[0243] 24 hours after treatment, electrocardiogram evaluation was performed, and peripheral blood was collected to detect myocardial injury biomarkers:

[0244] Troponin I (cTnI)

[0245] Creatine kinase-MB (CK-MB)

[0246] (2) Echocardiographic evaluation

[0247] At 4 weeks after treatment, a transthoracic echocardiogram was performed on the small pigs using a veterinary ultrasound system. The animals were in a light anesthetized state (inhalation of 1-1.5% isoflurane), and the right lateral position was taken.

[0248] Standard parasternal long-axis and short-axis cross-sectional images were obtained, and M-mode ultrasound measurements were taken: left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD).

[0249] Accordingly, left ventricular ejection fraction (LVEF), ventricular fractional shortening (LVFS) were calculated.

[0250] In addition, pulsed wave Doppler technology was used to measure the mitral valve orifice:

[0251] The peak velocity of rapid filling phase (E wave) and the peak velocity of atrial contraction phase (A wave) were measured. The ratio of E / A was calculated. Heart rate (HR) was calculated according to M-mode images. All images were saved digitally and analyzed offline using professional software.

[0252] (3) Myocardial fibrosis evaluation

[0253] At the 4th week after treatment, the animals were euthanized under deep anesthesia, the heart was quickly removed, washed with cold saline, and cut into 5 slices of about 5 mm thick from the apex to the base of the heart.

[0254] Each slice was placed in a 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution and incubated at 37°C in the dark for 20 minutes, then fixed in 4% paraformaldehyde.

[0255] Staining results:

[0256] Viable myocardium is red;

[0257] Infarct / fibrosis area does not stain and appears pale.

[0258] Each slice was photographed by a digital camera, and the following measurements were made using ImageJ software:

[0259] Infarct area, total left ventricular area.

[0260] The formula for calculating the percentage of fibrosis area is:

[0261] Fibrosis rate (%) = infarct area / total left ventricular area x 100%

[0262] The fibrosis rate of each heart was taken as the average value of each slice.

[0263] (4) Detection of cTnI and CK-MB

[0264] The specific enzyme-linked immunosorbent kit was used to detect the level of serum troponin I (cTnI), and the kit used was TNNI3 ELISA kit (ELK Biotechnology), and the detection steps were strictly performed according to the manufacturer's instructions.

[0265] At the same time, the species-specific kit provided by Coibo Company was used to detect the level of creatine kinase-MB (CK-MB).

[0266] After the successful establishment of the model in each experimental group, 0.2 mL of venous blood was collected for the detection of the above indicators.

[0267] (5) Optical imaging and electrophysiological evaluation

[0268] Before anesthesia, the pigs were treated with heparin (3000 U / kg) by intraperitoneal injection. Subsequently, the pigs were anesthetized with isoflurane and the hearts were rapidly excised and mounted on a Langendorff perfusion system. The hearts were perfused with cold (4°C) calcium-free Krebs buffer solution.

[0269] After 10 min of stable perfusion, blebbistatin was added to inhibit myocardial contraction, followed by sequential loading of Pluronic F127, the calcium indicator Rhod-2 AM and the membrane potential dye RH237.

[0270] The heart was placed in the optical imaging chamber with electrodes placed on the left ventricular apex, right atrium and the bottom of the perfusion chamber for grounding.

[0271] Optical and electrophysiological signals were recorded by:

[0272] sinus rhythm;

[0273] cardiac pacing (6 Hz);

[0274] calcium transient (Ca 2+ transient) signals.

[0275] Ventricular effective refractory period (VERP) was measured using S1-S2 stimulation protocol, which was achieved by shortening the S1-S2 interval. 50 Hz burst pacing was used to induce arrhythmias using increasing current (5-20 mA).

[0276] Action potential duration, depolarization / re-polarization process and calcium dynamics were analyzed by electrocardiogram and optical signal synchronization.

[0277] (6) In situ TUNEL apoptosis staining

[0278] At 24 h after treatment, the pigs were euthanized under deep anesthesia and the hearts were rapidly removed. The tissue from the border zone was embedded in OCT embedding medium and frozen sections of 5 μm thickness were prepared using a cryostat microtome.

[0279] Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed using a commercial apoptosis detection kit (Roche) according to the manufacturer's instructions.

[0280] Specific steps:

[0281] The sections were fixed with 4% paraformaldehyde for 15 min;

[0282] Permeabilized with 0.1% Triton X-100 / sodium citrate buffer;

[0283] Add TUNEL reaction solution, incubate at 37°C for 60 minutes in the dark;

[0284] Stain the nucleus with DAPI.

[0285] Image using fluorescence microscope, green signal is TUNEL positive cells, blue is the nucleus.

[0286] Randomly select five fields in each section, calculate the percentage of TUNEL positive nuclei in the total number of nuclei as the cell death index.

[0287] (7) Toxicity evaluation

[0288] At 24 hours after treatment, peripheral blood was collected through the auricular vein. The following serum indicators were measured using an automatic biochemical analyzer to evaluate kidney and liver function:

[0289] Uric acid (UA), urea (Urea), gamma-glutamyl transferase (γ-GT), aspartate aminotransferase (AST).

[0290] At the same time, the main organ tissues such as heart, liver, spleen, lung, and kidney were taken and fixed in 10% neutral buffered formalin, paraffin-embedded and sectioned (thickness 5 μm). Hematoxylin-eosin (H&E) staining was performed according to the standard procedure.

[0291] Observe the tissue morphology under a light microscope to evaluate whether there is structural damage or inflammatory cell infiltration to determine potential tissue toxicity.

[0292] (8) Transmission electron microscopy observation of cardiac mitochondria

[0293] On the 3rd day of treatment, the miniature pigs were euthanized under deep anesthesia, and myocardial tissue from the infarct edge area was taken. Immediately, about 1 mm 3 of tissue was cut and fixed with 2.5% glutaraldehyde at 4°C overnight.

[0294] Subsequently, 1% high osmotic acid post-fixation was performed for 1 hour, and the following treatments were performed in sequence:

[0295] Gradient ethanol dehydration;

[0296] Embedding with epoxy resin;

[0297] Ultrathin sectioning (thickness about 70 nm);

[0298] Double staining with uranyl acetate and lead citrate.

[0299] Finally, transmission electron microscopy was used for observation. Randomly select no less than five fields in each sample to evaluate the morphological structure of mitochondria.

[0300] (9) Western Blot analysis

[0301] On the 3rd day of treatment, myocardial tissue was collected from the peri-infarct region. Total protein was extracted by homogenizing the tissue with RIPA lysis buffer containing protease and phosphatase inhibitors.

[0302] After quantifying the total protein by BCA method, it was loaded for SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The following primary antibodies were used for incubation, respectively:

[0303] PGC-1a, COX IV, PINK1, TOMM20, GAPDH (as internal reference)

[0304] Then incubate with HRP-labeled secondary antibody and visualize by chemiluminescent substrate (ECL). All antibodies were purchased from Proteintech company.

[0305] (10) RNA sequencing and bioinformatics analysis

[0306] On the 3rd day of treatment, total RNA was extracted from the peri-infarct region, and TRIzol reagent was used for lysis. The integrity and quality of RNA were evaluated by RNA integrity value (RIN), ensuring RIN > 7.0.

[0307] Qualified samples were sent to Tsingke Biotechnology Co., Ltd. for library construction and high-throughput sequencing.

[0308] The obtained RNA sequencing data were used for: differential gene expression analysis; pathway enrichment analysis, mainly based on: KEGG (Kyoto Encyclopedia of Genes and Genomes), GSEA (Gene Set Enrichment Analysis).

[0309] To reveal the changes in treatment-related molecular mechanisms and signaling pathways.

[0310] (11) Myocardial protective effect of exosome microneedles encapsulating functional mitochondria in Bama mini-pig MI / R model

[0311] To evaluate the myocardial protective effect of P5CS or ATP5B-rich functional mitochondrial exosomes in different periods of myocardial ischemia-reperfusion (MI / R), we established an MI / R model in Bama mini-pigs and fixed mixed exosome microneedle patches (MN(MixEvs)) on the myocardial surface in the early ischemic period (n=6) Figure 8 Group A, Figure 8 Group B).

[0312] Ev(MTP5CS) showed significant cardioprotective effect during ischemia. P5CS is a key enzyme in arginine metabolic pathway, which can promote glutamine metabolism, maintain mitochondrial homeostasis, buffer energy fluctuations and enhance antioxidant defense. After MN(MixEvs) treatment, electrocardiogram(ECG) showed significant reduction in S-T segment elevation ( Figure 8 Mid-C, Figure 8 Mid-D), serum levels of cardiac troponin I(cTnI) and creatine kinase isoenzyme MB(CK-MB) decreased significantly ( Figure 8 Mid-E, Figure 9 Mid-A), suggesting that by regulating redox status and metabolic pathways, it effectively alleviated ischemia-induced myocardial cell damage.

[0313] Ev(MTATP5B) significantly improved cardiac function and tissue structure during reperfusion. ATP5B is the mitochondrial ATP synthase β subunit, which is a key component of the oxidative phosphorylation system, and is critical for ATP level recovery after reperfusion. The left ventricular systolic and diastolic function of MN(MixEvs) treatment group was significantly improved ( Figure 8 Mid-F- Figure 8 Mid-I, Figure 9 Mid-B), left ventricular ejection fraction(LVEF) increased, and infarct size decreased significantly ( Figure 8 Mid-J, Figure 8 Mid-K), indicating that ATP5B-rich mitochondria play a key role in inhibiting myocardial cell death and limiting ventricular remodeling.

[0314] Electrophysiological evaluation showed that MN(MixEvs) could restore action potential conduction and improve calcium ion regulation ability, significantly shorten the action potential duration(APD90) and calcium transient duration(CTD90) ( Figure 8 Mid-L, Figure 9 Mid-C- Figure 9 Mid-F), suggesting that it can reduce the risk of arrhythmia, and the mechanism may be related to the improvement of membrane potential stability. TUNEL staining further confirmed that MN(MixEvs) significantly reduced myocardial cell apoptosis ( Figure 8 Mid-M, Figure 9 Mid-G), supporting the direct cell protective effect of functional mitochondria through stabilizing membrane structure and metabolic balance.

[0315] Systemic toxicity evaluation showed that there were no abnormalities in serum liver and kidney function indicators in each group, and no obvious organ damage was found in histology ( Figure 9 Mid-H- Figure 9 Mid-L), indicating that the delivery system has good in vivo biocompatibility.

[0316] In summary, the present application first confirms in a large animal model that Ev(MTP5CS) and Ev(MTATP5B) delivered by exosomes exert a phased and complementary myocardial protective effect during the ischemia and reperfusion stages of MI / R. P5CS-rich mitochondria can alleviate oxidative stress and metabolic disorders during ischemia, while ATP5B-rich mitochondria can promote energy recovery and electrophysiological stability during the reperfusion period. This stage-specific mitochondrial intervention strategy provides a new mechanism basis and translational medicine potential for MI / R treatment.

[0317] (12) MN(MixEvs) exerts a cytoprotective effect by maintaining mitochondrial structure and function.

[0318] To explore the myocardial protection mechanism of MN(MixEvs) in MI / R, we systematically evaluated the structural and functional status of mitochondria in the myocardial tissue of Bama mini-pigs.

[0319] Transmission electron microscopy (TEM) results showed that the mitochondrial cristae structure was significantly improved in the MN(MixEvs) treatment group, and the outer membrane integrity was maintained ( Figure 10 Fig. 4A, Figure 10 Fig. 4B), indicating that it has a direct protective effect at the structural level. Mitochondrial structural damage is one of the important mechanisms of cell death induced by MI / R.

[0320] Protein analysis showed that PGC-1a expression was significantly up-regulated in the myocardial tissue of the treatment group, indicating enhanced mitochondrial biogenesis function; cytochrome oxidase IV (COX IV) expression increased, reflecting the recovery of oxidative phosphorylation function; PINK1 expression decreased, indicating reduced mitochondrial damage; and mitochondrial outer membrane marker TOMM20 expression recovered, indicating improved mitochondrial membrane structure ( Figure 10 Fig. 4C). These results indicate that MN(MixEvs) can enhance the mitochondrial quality control ability of myocardial cells, thereby improving their tolerance to MI / R stress.

[0321] RNA sequencing and KEGG pathway enrichment analysis showed that genes related to energy pathways such as oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and fatty acid metabolism were significantly up-regulated in the treatment group, indicating enhanced myocardial energy metabolism capacity. At the same time, mitochondrial autophagy (mitophagy) and PPAR signaling pathways were also significantly enriched, indicating that this strategy helps to eliminate damaged mitochondria and rebuild lipid metabolism homeostasis ( Figure 10 Fig. 4D).

[0322] GSEA enrichment analysis further confirmed that MN(MixEvs) can significantly activate metabolic and survival pathways such as TCA cycle, PI3K-Akt, and PPAR, while inhibiting stress and death pathways such as HIF-1, calcium signaling, apoptosis, and MAPK.Figure 10 The stabilization of membrane potential and the reduction of ROS level are particularly important for the inhibition of cardiomyocyte apoptosis during the reperfusion stage.

[0323] In summary, MN (MixEvs) protects cardiomyocytes through a multi-level mechanism: stabilizing mitochondrial structure, restoring metabolic function, promoting autophagy clearance, and regulating multiple cell protection signaling pathways, fully verifying the precise treatment potential of "functional mitochondria delivery" in MI / R intervention.

[0324] In summary, the present application starts from the heterogeneity of cardiomyocyte subpopulations and first discovers that HEY1⁺ cardiomyocytes (HEY1⁺ CMs) can maintain stable mitochondrial function under ischemic stress. Based on this, we constructed functional exosomes rich in P5CS or ATP5B, realizing the differential delivery of functional mitochondria during the ischemic and reperfusion stages. Combined with the dynamic changes in copper / iron ion concentration in the myocardial microenvironment, we designed MN (MixEvs) microneedle patches to precisely control the phased release of functional exosomes.

[0325] In animal models, this strategy effectively alleviates myocardial ischemia-reperfusion injury, restores cardiac function, and improves myocardial electrophysiological stability without observing obvious toxic side effects. This scheme integrates the functional characteristics of cell subtypes, biological response materials, and mitochondrial targeted intervention means to construct a myocardial protection system with time specificity and targeting.

[0326] This research provides a new treatment paradigm for the precise treatment of myocardial infarction and shows strong clinical transformation potential in the combined intervention around different stages of the PCI operation process.

[0327] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A microneedle patch, characterized in that, It includes: The needle body, the base, and the barrier layer located on the back of the base, wherein the needle body is located on the base, and the needle body and the base together are arranged in an array on the barrier layer; The needle body comprises engineered exosome 1, a copper-responsive active peptide, and a dopamine-modified needle matrix; the copper-responsive active peptide is covalently cross-linked with a quinone group formed by the oxidation of the thiol group to obtain a cross-linked product; and the exosome 1 is embedded in the cross-linked product formed by the copper-responsive active peptide and the dopamine-modified needle matrix; the engineered exosome 1 contains mitochondria with high P5CS expression; and the engineered exosome 1 is derived from HEY1-positive cardiomyocytes with high P5CS expression; the amino acid sequence of the copper-responsive active peptide is: SH-(GGH)n-SH, where n is 3-5; The pedicle comprises engineered exosome 2, an iron-responsive active peptide, and a dopamine-modified pedicle matrix. The iron-responsive active peptide is covalently cross-linked with a quinone group formed by the oxidation of dopamine to obtain a cross-linked product. The exosome 2 is embedded in the cross-linked product formed by the iron-responsive active peptide and the dopamine-modified needle matrix. The engineered exosome 2 contains mitochondria with high ATP5B expression. The engineered exosome 2 is derived from HEY1-positive cardiomyocytes with high ATP5B expression. The amino acid sequence of the iron-responsive active peptide is: SH-(HG)n-SH, where n is 3-5. The barrier layer comprises a hydrogel material.

2. The microneedle patch according to claim 1, characterized in that, The portion connected to the bottom of the needle body is the upper part of the base, and the bottom part away from the needle body is the lower part of the base; wherein, the combination of the upper part of the base and the needle body forms a frustum or a cone. The diameter of the bottom end of the frustum or cone is 100-300 μm; the height of the frustum or cone is 100-500 μm; the array density is 100-1000 needles / square centimeter; and the diameter of the top end of the frustum or cone is less than 80 μm.

3. The microneedle patch according to claim 1, characterized in that, The needle matrix is ​​selected from at least one of polycaprolactone-block-polyacrylic acid, hyaluronic acid, polyvinyl alcohol, polycarboxymethyl cellulose, polyethylene glycol, polylactic acid, polyvinylpyrrolidone, chondroitin sulfate, polylactic acid-polyglycolic acid copolymer, silk protein, cyclodextrin, zinc hyaluronic acid, and gelatin. The substrate matrix is ​​selected from at least one of the following: sodium hyaluronate-maleimide, hydrazylated hyaluronic acid, aldehyde-modified hyaluronic acid, hyaluronic acid-catechol, polyethylene glycol-maleimide, polyethylene glycol diacrylate, gelatin methacrylamide, gelatin-maleimide, aldehyde-modified chitosan, chitosan-maleimide, polyvinyl alcohol-catechol, aldehyde-modified polyvinyl alcohol, polycarboxymethyl cellulose-maleimide, aldehyde-modified polycarboxymethyl cellulose, aldehyde-modified alginate, hydrazylated alginate, alginate-catechol, silk fibroin-catechol, and collagen-maleimide.

4. The microneedle patch according to claim 3, characterized in that, The needle matrix is ​​selected from polycaprolactone-block-polyacrylic acid.

5. The microneedle patch according to claim 1, characterized in that, The material of the barrier layer is selected from PCA hydrogel, sodium alginate hydrogel, hyaluronic acid hydrogel, chitosan hydrogel, cellulose hydrogel, collagen hydrogel, polyvinyl alcohol hydrogel, polyethylene glycol hydrogel, poly(N-isopropylacrylamide) hydrogel, gelatin / PEG composite hydrogel, or chitosan / polyacrylic acid hydrogel.

6. The method for preparing the microneedle patch according to any one of claims 1-5, characterized in that, It includes the following steps: mixing the engineered exosome 1, copper-responsive active peptide and dopamine-modified needle matrix in a mass ratio of (1-5):(3-15):(20-100) to prepare a pregel solution; mixing the engineered exosome 2, iron-responsive active peptide and dopamine-modified base matrix in the following mass ratio: (5-25):(1-8):100 to prepare a precursor solution; The pregel solution is added to the microneedle mold; before it solidifies, the precursor solution is added; after placement, the hydrogel material used to prepare the barrier layer is coated on the surface of the substrate. After curing, the microneedle patch is demolded.

7. The method for preparing the microneedle patch according to claim 6, characterized in that, The barrier layer is cross-linked and cured by light irradiation. The hydrogel material used to prepare the barrier layer includes: polyethylene glycol diacrylate, acrylic acid and carboxylated cellulose nanocrystals prepared in a mass ratio of 2–8 : 1–4 : 0.3–2.0, and 0.5–2.0 wt% photoinitiator. The photo-irradiation crosslinking curing is performed by irradiating with ultraviolet light for 10-20 minutes.

8. The method for preparing the microneedle patch according to claim 6, characterized in that, The engineered exosomes 1 and 2 were prepared by the following method: First, cardiomyocytes overexpressing HEY1 were prepared; then, the cardiomyocytes overexpressing HEY1 were transfected with a lentiviral vector carrying P5CS to obtain HEY1-positive cardiomyocytes with high P5CS expression; the cardiomyocytes overexpressing HEY1 were transfected with a lentiviral vector carrying ATP5 to obtain HEY1-positive cardiomyocytes with high ATP5 expression; the lentiviral vector was equipped with FCCPs to facilitate mitochondrial entry into exosomes; The P5CS-high-expressing HEY1-positive cardiomyocytes and the ATP5-high-expressing HEY1-positive cardiomyocytes were treated with mitochondrial uncoupling agents to induce the release of mitochondrial components into exosomes. Then, exosomes were extracted to obtain exosome 1 containing mitochondria with high expression of P5CS and exosome 2 containing mitochondria with high expression of ATP5.

9. A medicine for preventing or treating a disease, characterized in that, It includes the microneedle patch as described in any one of claims 1-5 or the microneedle patch prepared by the preparation method as described in any one of claims 6-8: The disease is selected from: myocardial ischemia and / or reperfusion injury, or ischemic heart disease with mitochondrial dysfunction / oxidative stress as the main feature; The treatment options are selected from: post-infarction functional recovery and anti-remodeling, PCI, CABG, or valve surgery.

10. The use of the microneedle patch according to any one of claims 1-5 or the microneedle patch prepared by the preparation method according to any one of claims 6-8 in the preparation of drugs for the prevention or treatment of myocardial ischemia and / or reperfusion injury, characterized in that, The drug was administered preoperatively.

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