NO in-situ continuous delivery system of targeted myocardial cell mitochondria as well as preparation method and application of NO in-situ continuous delivery system

Abstract

The invention provides an NO in-situ continuous delivery system of targeted myocardial cell mitochondria as well as a preparation method and application of the NO in-situ continuous delivery system, and belongs to the technical field of pharmaceutical preparations. The NO in-situ continuous delivery system of the targeted myocardial cell mitochondria, provided by the invention, comprises a NO nano-delivery carrier of the targeted myocardial cell mitochondria and an NO microneedle patch prepared from the NO nano-delivery carrier of the targeted myocardial cell mitochondria. The NO nano delivery carrier is prepared by adopting ZIF-90 to bear NO and modifying triphenylphosphine, the mitochondrial targeting delivery function is realized, and the loading efficiency of L-arginine reaches 40%. The NO nano delivery carrier and the hydrogel are combined to form the microneedle patch, and NO can be stably supplied to myocardial cells. The invention has unique advantages, can realize continuous administration and accurate positioning, significantly reduces toxic and side effects, effectively eliminates excessive ROS, powerfully maintains ventricular strength and recovers electrical conduction, shows good biocompatibility, and provides a potential solution for clinical application of myocardial ischemia diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to an in situ sustained delivery system of NO targeting myocardial cell mitochondria, and a preparation method and application thereof. Background Art

[0002] NO (Nitric oxide) is a vital gas signaling molecule in the cardiovascular system. It plays an indispensable role in maintaining normal vascular physiological functions and finely regulating vascular homeostasis. Specifically, NO can reduce myocardial ischemia-reperfusion injury by relaxing vascular tension, inhibiting platelet aggregation, and regulating inflammatory responses. It can also inhibit the activity of complex I in the mitochondrial electron transport chain, thereby limiting the generation of mitochondrial ROS (reactive oxygen species), further preventing the opening of the mitochondrial permeability transition pore (mPTP), reducing the release of cytochrome c and apoptosis of cardiomyocytes. However, NO is like a "double-edged sword". Its efficacy shows an extremely strict dose-dependency. Once the dose is too high, it will induce cell apoptosis and produce toxicity. In addition, when nitric oxide is released systemically, it will also cause a series of side effects such as decreased blood pressure and accelerated heart rate. These unfavorable factors seriously limit the scope of application and effect of NO in clinical treatment.

[0003] Myocardial infarction is one of the key factors leading to death from cardiovascular diseases. In the case of myocardial infarction, timely reperfusion therapy can effectively prevent myocardial cell necrosis to a certain extent. However, the process of blood reperfusion will induce myocardial cell death and fibrosis, which will aggravate and further amplify the degree of myocardial ischemia damage, and eventually form a complex and difficult condition of myocardial ischemia-reperfusion injury. At present, there are many clinical methods for the treatment of myocardial infarction, including vascular bypass and coronary stent implantation. These methods can indeed restore cardiac perfusion relatively quickly and effectively, but the ischemic process often causes irreversible damage to myocardial cells, and subsequent blood reperfusion will make the situation worse and further aggravate this damage. Taking nitroglycerin as an example, it can be converted and release NO inside the cell, thereby achieving the physiological effect of relaxing vascular smooth muscle, thereby achieving the therapeutic effect of dilating blood vessels and increasing coronary blood perfusion. However, the current oral and intravenous delivery of NO lacks targeting, and the effect of improving the ischemic microenvironment and inhibiting cell apoptosis is unsatisfactory. Currently, clinical treatment still lacks feasible and effective NO targeted delivery preparations. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an in situ sustained delivery system of NO targeting myocardial cell mitochondria, and its preparation method and application, to achieve targeted delivery of NO in mitochondria through a nano targeted delivery system and a microneedle patch, and to provide an effective therapeutic preparation for improving myocardial ischemia and related symptoms.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a NO nano delivery carrier targeting myocardial cell mitochondria, comprising a nano carrier ZIF-90, L-arginine and a surface modification molecule triphenylphosphine.

[0007] The invention provides a method for preparing a NO nano delivery carrier targeting myocardial cell mitochondria. The preparation method comprises the following steps: loading L-arginine on a nano carrier ZIF-90 by an electrostatic adsorption method to obtain L-Arg@ZIF-90 nanoparticles; and modifying triphenylphosphine molecules on the surface of the L-Arg@ZIF-90 nanoparticles by an EDC / NHS reaction to obtain a NO nano delivery carrier L-Arg@ZIF-90-TPP targeting myocardial cell mitochondria.

[0008] Preferably, the mass ratio of L-arginine to nanocarrier ZIF-90 is 1:(0.5-8).

[0009] Preferably, the mass ratio of the L-Arg@ZIF-90 nanoparticles, EDC, NHS and triphenylphosphine is 1:(1-2):(0.5-1):(2-3).

[0010] The present invention also provides the use of the aforementioned NO nano-delivery vector targeting myocardial cell mitochondria or the aforementioned preparation method in the preparation of a drug, wherein the drug is any one or two of the following drugs:

[0011] 1) The drug is a drug for preventing and / or treating myocardial ischemia-reperfusion injury;

[0012] 2) The drug is a drug that inhibits the production of reactive oxygen species in cell mitochondria.

[0013] The present invention also provides a NO microneedle patch based on a nano delivery carrier, wherein the microneedle patch comprises the aforementioned NO nano delivery carrier targeting myocardial cell mitochondria and a gel matrix.

[0014] The present invention also provides a method for preparing a NO microneedle patch based on a nano delivery carrier, the preparation method comprising the following steps:

[0015] 1) GelMA, PPY and ammonium persulfate are cross-linked to obtain GelMA-PPY;

[0016] 2) GelMA-PPY, GelMA, PEGDA, and polydopamine were mixed and cross-linked to obtain a GelMA-PPY-PDA gel matrix;

[0017] 3) L-Arg@ZIF-90-TPP nanoparticles were added to the photoinitiator, and then mixed with the GelMA-PPY-PDA gel matrix, and photocured to obtain the NO microneedle patch encapsulating the L-Arg@ZIF-90-TPP nanoparticles.

[0018] Preferably, the mass ratio of GelMA, PPY and ammonium persulfate in step 1) is 75:(5-30):(10-50);

[0019] Preferably, in the step 2), the feed mass ratio of GelMA-PPY, GelMA, PEGDA and polydopamine is (4000-6000):(40-60):(80-120):1.

[0020] Preferably, in the step 3), the mass ratio of the GelMA-PPY-PDA gel matrix to the L-Arg@ZIF-90-TPP nanoparticles is (1-10):1.

[0021] The present invention also provides a NO microneedle patch based on a nano-delivery carrier, or the use of the aforementioned preparation method in preparing a pharmaceutical preparation, wherein the pharmaceutical preparation is any one or more of the following pharmaceutical preparations:

[0022] 1) The pharmaceutical preparation is a pharmaceutical preparation for removing excess ROS in myocardial tissue after myocardial infarction;

[0023] 2) The pharmaceutical preparation is a pharmaceutical preparation for preventing and / or treating myocardial ischemia-reperfusion injury;

[0024] 3) The pharmaceutical preparation is a pharmaceutical preparation for preventing and / or treating arrhythmia caused by myocardial ischemia;

[0025] 4) The pharmaceutical preparation is a pharmaceutical preparation for improving the conduction of electrical signals in myocardial tissue.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a NO in situ sustained delivery system targeting myocardial cell mitochondria, and a preparation method and application thereof. In the present invention, the NO nano-delivery carrier targeting myocardial cell mitochondria uses ZIF-90 as a NO-bearing matrix, and triphenylphosphine is carefully modified on its surface and L-arginine is adsorbed, making full use of the unique mitochondrial targeting properties of triphenylphosphine and the performance of ZIF-90 that degrades in response to the mitochondrial environment, thereby achieving NO targeted delivery in the mitochondrial site, and the NO nano-delivery carrier prepared by the present invention can have a loading efficiency of up to 40% for L-arginine, thereby improving the delivery efficiency of NO.

[0028] On this basis, the present invention also provides a NO microneedle patch based on a nano-delivery carrier, which organically integrates the NO nano-delivery carrier with a gel matrix, so as to continuously and stably supply NO to myocardial cells. Compared with myocardial injection by a syringe, the microneedle patch of the present invention can keep the injection site in the myocardium rather than the heart cavity, achieve in situ continuous drug administration, and do not affect other normal organs; compared with intravenous injection, the microneedle patch of the present invention is positioned for drug administration, effectively preventing the impact of NO on the whole body system, and reducing the toxic and side effects that NO may cause to other normal tissues. In addition, the NO microneedle patch provided by the present invention can effectively remove excess ROS in myocardial tissue and give full play to the positive role of anti-oxidation. The NO microneedle patch of the present invention not only has the basic function of maintaining the mechanical strength of the ventricle, but also has good electrical conductivity, can effectively restore the electrical conduction signals in damaged myocardial tissue, and has extremely critical therapeutic value and far-reaching significance for improving arrhythmias and other diseases. It has been verified that the NO microneedle patch based on the nano-delivery carrier prepared by the present invention exhibits good biocompatibility, does not affect liver function and kidney function, and does not cause pathological damage to other normal tissues, laying a solid and reliable foundation for its clinical application in diseases such as myocardial ischemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the infrared spectrum of L-Arg@ZIF-90 nanoparticles.

[0030] Figure 2 This is the particle size distribution diagram of L-Arg@ZIF-90 nanoparticles.

[0031] Figure 3 TEM image of L-Arg@ZIF-90 nanoparticles, scale bar 200nm.

[0032] Figure 4 The cytotoxic effects of different concentrations of L-Arg@ZIF-90 nanoparticles on H9C2 cells.

[0033] Figure 5 This is the particle size distribution diagram of L-Arg@ZIF-90-TPP.

[0034] Figure 6 This is the XRD spectrum of L-Arg@ZIF-90-TPP.

[0035] Figure 7 This is the infrared spectrum of L-Arg@ZIF-90-TPP.

[0036] Figure 8 TEM image of L-Arg@ZIF-90-TPP, scale 200nm.

[0037] Fig. 9 This is the cytotoxicity result of L-Arg@ZIF-90-TPP on H9C2 cells, n=6.

[0038] Fig.10 This is the current-potential diagram of GelMA-PPY-PDA hydrogel.

[0039] Fig.11 Impedance diagram of GelMA-PPY-PDA hydrogel.

[0040] Fig.12 This is the H NMR spectrum of GelMA-PPY-PDA.

[0041] Fig.13 Microscope photograph of the microneedle patch doped with L-Arg@ZIF-90-TPP nanoparticles, scale bar: 200μm.

[0042] Fig.14 This is an experimental diagram of the co-localization of L-Arg@ZIF-90-TPP nanoparticles in neonatal rat cardiomyocytes. Scale bar: 20 μm, where red fluorescence indicates mitochondria, blue indicates cell nuclei, and green fluorescence comes from FITC-labeled L-Arg@ZIF-90-TPP nanoparticles.

[0043] Fig.15 Cardiac fluorescence imaging of the microneedle patch doped with L-Arg@ZIF-90-TPP nanoparticles at 1, 2, and 3 weeks after transplantation into the hearts of mice with myocardial ischemia-reperfusion injury. ns indicates no significant difference, *P < 0.05.

[0044] Fig.16These are the echocardiographic data of improvement of cardiac function in mice with myocardial ischemia-reperfusion injury treated with microneedle patches 3 weeks later. Sham is a sham-operated healthy group, I / RI is a model group, I / RI+GelMA-PPY-PDA is a gel treatment group prepared in Example 3, I / RI+L-Arg@ZIF-90-TPP+GelMA-PPY-PDA is a microneedle patch treatment group prepared in Example 4, I / RI+L-Arg@ZIF-90-TPP+GelMA is a microneedle treatment group doped with L-Arg@ZIF-90-TPP nanoparticles, and I / RI+L-Arg+GelMA-PPY is a microneedle treatment group doped with L-Arg; ns indicates no significant difference, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0045] Fig.17 The figure shows the effect of NO microneedle patch on liver and renal function indicators of mice with myocardial ischemia-reperfusion injury after 3 weeks of treatment. Sham is a sham-operated healthy group, I / RI is a model group, I / RI+GelMA-PPY-PDA is a gel treatment group prepared in Example 3, I / RI+L-Arg@ZIF-90-TPP+GelMA-PPY-PD A is a microneedle patch treatment group prepared in Example 4, I / RI+L-Arg@ZIF-90-TPP+GelMA is a microneedle treatment group doped with L-Arg@ZIF-90-TPP nanoparticles, and I / RI+L-Arg+GelMA-PPY is a microneedle treatment group doped with L-Arg@ZIF-90-TPP nanoparticles; ns indicates no significant difference. DETAILED DESCRIPTION

[0046] The invention provides a NO nano delivery carrier targeting myocardial cell mitochondria, comprising a nano carrier ZIF-90, L-arginine and a surface modification molecule triphenylphosphine.

[0047] In the present invention, the L-arginine is loaded on the surface of the nanocarrier ZIF-90 particles by electrostatic adsorption, L-arginine (L-Arg) exists as a NO donor, and triphenylphosphine molecules are modified on the surface of ZIF-90 to target mitochondria.

[0048] The present invention also provides a method for preparing a NO nano delivery carrier targeting myocardial cell mitochondria, which specifically comprises the following steps: L-arginine is loaded on the surface of the nanocarrier ZIF-90 by electrostatic adsorption to obtain L-Arg@ZIF-90 nanoparticles. Triphenylphosphine molecules are modified on the surface of the L-Arg@ZIF-90 nanoparticles by EDC / NHS reaction to obtain the NO nano delivery carrier L-Arg@ZIF-90-TPP targeting myocardial cell mitochondria.

[0049] The present invention does not particularly limit the source of the nanocarrier ZIF-90, which can be synthesized by itself or purchased commercially. As an embodiment, the nanocarrier ZIF-90 can be prepared by self-assembly of zinc salt and imidazole molecules in a solvent. The zinc salt includes one or more of zinc acetate, zinc nitrate, zinc chloride, zinc sulfate or their hydrates. Preferably, the zinc salt is zinc acetate or zinc acetate hydrate. Further preferably, the zinc salt is zinc acetate. The imidazole molecules include 2-imidazole carboxaldehyde. The solvent includes ultrapure water, methanol, ethanol, DMF (N, N-dimethylformamide). Preferably, the solvent is DMF. As an embodiment, the nanocarrier ZIF-90 is prepared by self-assembly of zinc acetate and 2-imidazole carboxaldehyde in DMF. The feed ratio of zinc acetate, 2-imidazole carboxaldehyde and DMF is preferably 1 mg: 0.5-10 mg: 0.5-10 mL, more preferably 1 mg: 1-8 mg: 0.8-8 mL, further preferably 1 mg: 2-6 mg: 1.5-6 mL, and further preferably 1 mg: 4 mg: 4 mL. The temperature of the self-assembly is preferably 20-120° C., more preferably 50-80° C., and most preferably 70° C.

[0050] In the present invention, L-arginine is loaded on the surface of the nanocarrier ZIF-90 by electrostatic adsorption, and L-arginine is adsorbed on the surface of the nanocarrier ZIF-90 in a solvent to obtain L-Arg@ZIF-90 nanoparticles. The solvent includes ultrapure water, methanol, ethanol, and DMF. Preferably, the solvent is ultrapure water. The mass ratio of L-arginine to the nanocarrier ZIF-90 is 1: (0.5-8), preferably 1: (1-6), and further preferably 1: (1-4). In the present invention, after L-arginine and the nanocarrier ZIF-90 are mixed in a mass ratio, stirred for reaction, centrifuged, and washed to obtain L-Arg@ZIF-90 nanoparticles. The stirring is performed by magnetic stirring. The stirring speed is 400-800rpm / min, preferably 500-700rpm / min, and further preferably 600rpm / min. The stirring time is 0.5-2h, preferably 1h. The centrifugal speed is 8000-12000rpm / min, preferably 9000-11000rpm / min, and more preferably 10000rpm / min. The centrifugal time is 5-20min, preferably 8-15min, and more preferably 10-12min. In the present invention, triphenylphosphine molecules are modified on the surface of L-Arg@ZIF-90 nanoparticles by EDC / NHS reaction, so that L-Arg@ZIF-90 nanoparticles have mitochondrial targeting. The EDC / NHS reaction uses EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) as crosslinkers and stabilizers, respectively, to bind triphenylphosphine molecules to the surface of L-Arg@ZIF-90 nanoparticles. The mass ratio of the L-Arg@ZIF-90 nanoparticles, EDC, NHS and triphenylphosphine is 1: (1-2): (0.5-1): (2-3). Preferably it is 1: (1.1-1.4): (0.6-0.9): (2.4-2.8). In the present invention, EDC and triphenylphosphine are subjected to a binding reaction in a buffer. The buffer is preferably a MES buffer. The pH of the buffer is 3.5-5.5, preferably 4.0-5.0, and more preferably 4.5. The binding reaction time is 12-20 min, preferably 15-18 min. NHS is then added as a stabilizer to stabilize the reaction product of EDC and triphenylphosphine. The stabilization reaction is carried out in a buffer, and the buffer is preferably a PBS buffer. The pH of the buffer is 7.0-8.0, preferably 7.2-7.5. After the stabilization reaction is completed, L-Arg@ZIF-90 nanoparticles are added and stirred to carry out a surface modification reaction. After centrifugation, the precipitate is washed to obtain L-Arg@ZIF-90-TPP nanoparticles. The stirring is performed by magnetic stirring.The stirring speed is 200-600rpm / min, preferably 300-500rpm / min, and more preferably 400rpm / min. The stirring time is 15-60min, preferably 30min. The centrifugal speed is 5000-10000rpm / min, preferably 6000-9000rpm / min, and more preferably 7000-8000rpm / min. The centrifugal time is 5-20min, preferably 8-15min, and more preferably 10-12min. The washing is preferably washed with ultrapure water. The loading efficiency of the ZIF-90 nanocarrier prepared by the present invention for L-arginine can be as high as 40%, which improves the delivery efficiency of NO.

[0051] In the present invention, there is also provided an application of a NO nano delivery carrier targeting myocardial cell mitochondria and a preparation method thereof in the preparation of a drug, and the drug can be used to prevent and / or treat myocardial ischemia-reperfusion injury. The drug can also inhibit the generation of ROS (reactive oxygen) in cell mitochondria. The drug can target mitochondria and aggregate, and utilize the ATP-responsive degradation of ZIF-90 to release L-arginine, which is catalyzed by nitric oxide synthase to generate NO in the presence of O2 and NADPH, and plays a role in preventing and / or treating myocardial ischemia-reperfusion injury, as well as preventing and / or treating arrhythmias caused by myocardial ischemia. The NO produced by the drug can regulate the activity of the cell mitochondrial respiratory chain, affect the activity and function of the cell mitochondrial respiratory chain complex, metabolic enzymes, and mitochondrial-related proteins, and inhibit the generation of ROS. The cells include, but are not limited to, myocardial cells, as well as neuronal cells, tumor cells, macrophages, stem cells, etc. The drug can be prepared into various mitochondrial targeting preparations, including but not limited to microneedle patches, injections, liposomes, etc.

[0052] In the present invention, a NO microneedle patch based on a nano delivery carrier is provided, and the microneedle patch includes the aforementioned NO nano delivery carrier targeting myocardial cell mitochondria and a gel matrix. In the present invention, the gel matrix is ​​preferably a gel matrix obtained by cross-linking a photosensitive biomaterial under the action of a photoinitiator and curing. The photosensitive biomaterial includes but is not limited to GelMA (methacrylic acid acylated gelatin), HAMA (methacrylic acid esterified hyaluronic acid) and ALGMA (methacrylic acid acylated sodium alginate). As an embodiment, the photosensitive biomaterial is selected from GelMA. The photoinitiator includes LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphinate), lrgacure 2959, preferably LAP.

[0053] The present invention also provides a method for preparing a NO microneedle patch based on a nano delivery carrier, the preparation method comprising the following steps: 1) GelMA, PPY, and ammonium persulfate are cross-linked to obtain GelMA-PPY; 2) GelMA-PPY, GelMA, PEGDA, and polydopamine are mixed and cross-linked to obtain a GelMA-PPY-PDA gel matrix; 3) L-Arg@ZIF-90-TPP nanoparticles are added to a photoinitiator, and then mixed with the GelMA-PPY-PDA gel matrix, and photocured to obtain a NO microneedle patch encapsulating L-Arg@ZIF-90-TPP nanoparticles. The present invention has no special requirements for the source of the GelMA, which can be obtained through commercial channels, or prepared by a preparation method well known to those skilled in the art, that is, prepared from methacrylic anhydride and gelatin. The step 1) is to sequentially add GelMA, pyrrole (PPY), and ammonium persulfate to dilute hydrochloric acid, stir magnetically at room temperature, add NaOH to adjust the pH to neutral, and precipitate with anhydrous ethanol, and dry to obtain GelMA-PPY. The molecular weight of the GelMA is preferably 100-200 kDa. The concentration of the dilute hydrochloric acid is 0.1-0.5 mol / L, preferably 0.2-0.3 mol / L. The mass ratio of the GelMA, PPY, and ammonium persulfate is 75: (5-30): (10-50), preferably 75: (10-20): (20-40), and more preferably 75: (11-15): (25-35). The GelMA-PPY-PDA gel matrix in step 2) is formed by mixing and cross-linking the GelMA-PPY obtained in step 1) with GelMA, PEGDA (polyethylene glycol diacrylate), and polydopamine in an aqueous solution. The mass ratio of the GelMA-PPY, GelMA, PEGDA and polydopamine is (4000-6000): (40-60): (80-120): 1, preferably (4500-5500): (45-55): (90-110): 1, and more preferably (4800-5200): (48-52): (95-105): 1. The present invention also provides a method for preparing polydopamine, specifically: PDA is dissolved in acidic MBA (N, N-methylenebisacrylamide) under nitrogen protection, stirred in an oil bath in the dark, freeze-dried, and stored at -20°C. The pH of the acidic MBA is 5.0-7.0, preferably 6.0. The concentration of the PDA is 60-70 mg / ml, preferably 64-68 mg / ml. The temperature of the oil bath is 40-50°C, preferably 45°C. The stirring reaction is carried out for 60 to 80 hours, preferably 70 to 75 hours.Step 3) is to dissolve L-Arg@ZIF-90-TPP nanoparticles in a photoinitiator solution, mix with GelMA-PPY-PDA gel matrix, stir at room temperature, and after fully dissolved, pour into a microneedle mold, perform vacuum / centrifugal defoaming operation, and place in an oven for drying. The mass volume fraction of the photoinitiator is 0.20-0.30%, preferably 0.25%. The stirring time is 0.5-3h, preferably 1-2h. The drying temperature is 30-80°C, preferably 35-65°C. The number of drying times is 1-3 times, preferably 2 times. The drying time for each time is 3-7h, preferably 4-6h. The light curing wavelength is 405nm. The mass ratio of GelMA-PPY-PDA gel matrix to L-Arg@ZIF-90-TPP nanoparticles in the NO microneedle patch is (1-10):1, preferably (4-8):1, and more preferably (5-7):1. In the present invention, L-Arg@ZIF-90-TPP nanoparticles are randomly distributed on the needle tips of the NO microneedle patch made of the GelMA-PPY-PDA gel matrix.

[0054] The present invention also provides an application of a NO microneedle patch based on a nano delivery carrier and a preparation method thereof in the preparation of a pharmaceutical preparation. The NO microneedle patch prepared by the present invention has good biocompatibility and does not cause toxic effects on normal organs. The pharmaceutical preparation prepared by the NO microneedle patch can inhibit the excessive production of ROS in myocardial tissue of myocardial infarction. The pharmaceutical preparation can also prevent and / or treat myocardial ischemia-reperfusion injury, and prevent and / or treat arrhythmia caused by myocardial ischemia. In addition to having the function of maintaining the mechanical strength of the ventricle, the pharmaceutical preparation of the present invention also has electrical conductivity, can restore the electrical conduction signal in the damaged myocardial tissue, and play a role in improving the conduction of electrical signals in the myocardial tissue.

[0055] In the examples of the present invention, all data were statistically analyzed using software Orign and GraphPad Prism 9.0, and the data were expressed in the form of mean ± standard deviation.

[0056] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0057] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0058] The technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0059] Example 1

[0060] Preparation of L-Arg@ZIF-90 nanoparticles

[0061] Preparation of ZIF-90: Dissolve Zn(CH3COOH)2·2H2O in DMF to prepare a 0.1 mol / L solution for later use. Dissolve 2-imidazole carboxaldehyde in DMF to prepare a 0.2 mol / L solution for later use. Mix the two solutions, stir magnetically at 500 rpm / min for 5 minutes, add 10 mL of DMF, stir magnetically at 500 rpm / min for 20 minutes at 70°C, centrifuge at 1000 rpm / min for 20 minutes, discard the supernatant, wash once with DMF, wash twice with anhydrous ethanol, and freeze-dry to obtain ZIF-90, which is stored at 4°C.

[0062] L-Arg was dissolved in ultrapure water to prepare a solution with a concentration of 10 mg / mL for later use. ZIF-90 was fully dissolved in ultrapure water by ultrasonication for 15 minutes to prepare a solution with a concentration of 10 mg / mL for later use. ZIF-90:L-Arg was mixed in a mass ratio of 1:2, 2:2, 4:2, 8:2, 12:2, and 16:2, and reacted at 600 rpm / min magnetic stirring for 1 hour, and then centrifuged at 10000 rpm / min for 10 minutes. The supernatant was discarded and washed twice with ultrapure water to obtain L-Arg@ZIF-90 nanoparticles.

[0063] L-Arg@ZIF-90 nanoparticles were co-incubated with H9C2 cells for 24 h, and their cytotoxicity was detected by MTT assay.

[0064] The results are as follows Figure 1 , 2 , 3 and 4 show that L-Arg was successfully loaded into the ZIF-90 nanocarriers formed at different ZIF-90:L-Arg ratios. The infrared spectrum results show that the infrared characteristic peak of L-Arg exists inside L-Arg@ZIF-90. It has been tested that the loading efficiency of ZIF-90 nanocarriers for L-arginine reaches 40%. Its particle size is between 100 and 200 nm. Through the MTT cytotoxicity experiment, it is clear that L-Arg@ZIF-90 nanoparticles will not significantly inhibit the survival rate of H9C2 cells, indicating that L-Arg@ZIF-90 has low cardiomyocyte toxicity.

[0065] Example 2

[0066] Preparation of mitochondria-targeted L-Arg@ZIF-90-TPP nanoparticles

[0067] Use ultrapure water to prepare 25 mL of 0.05 mol / L MES (2-(N-morpholine)ethanesulfonic acid) buffer, take 250 mg of triphenylphosphine (TPP) and 116 mg of EDC and dissolve them in 23 mL of MES buffer, adjust the pH to 4.5 with dilute hydrochloric acid (0.1 M), react for 15 minutes, then add 69 mg of NHS to the above solution, adjust the pH to 7.2-7.5 with PBS, add 96 mg of L-Arg@ZIF-90, stir magnetically at 400 rpm / min for 0.5 h, let stand for 1.5 h, and finally centrifuge at 7000 rpm / min for 10 min. Wash twice with ultrapure water to obtain L-Arg@ZIF-90-TPP nanoparticles.

[0068] The results are as follows Figure 5 , 6 , 7, 8 and 9 show that the particle size of L-Arg@ZIF-90-TPP nanoparticles is within 200nm and has characteristic peaks of TPP and ZIF-90 through transmission electron microscopy and infrared spectroscopy. Through MTT cytotoxicity experiment, the survival rate of H9C2 cells decreases with the increase of L-Arg@ZIF-90-TPP concentration, indicating that L-Arg@ZIF-90-TPP has low cardiomyocyte toxicity.

[0069] Example 3

[0070] Preparation of GelMA-PPY-PDA microneedle patch

[0071] Preparation of polydopamine: Adjust the pH of deionized water / ethanol (V / V=4:3) solution to 6 with 5 mol HCl, add 500 mg MBA to a final concentration of 70.1 mg / ml. After dissolution, add 475 mg dopamine to the solution under nitrogen protection, stir in a 45°C oil bath in the dark for 3 days under nitrogen protection, and obtain polydopamine after freeze-drying, which is stored at -20°C for future use.

[0072] Preparation of GelMA-PPY: 75 mg of GelMA was completely dissolved in 25 ml of 0.1 mol HCl and stirred in a round-bottom flask. 12 mg of PPY was added thereto, and after dissolution, 30 mg of ammonium persulfate was added to the reaction medium and stirred continuously at room temperature overnight. The grafted polymer was neutralized with NaOH and precipitated with excess anhydrous ethanol, and dried at room temperature in the atmosphere to obtain the product GelMA-PPY.

[0073] Preparation of GelMA-PPY-PDA gel matrix: Prepare a 4.12% GelMA aqueous solution and set aside. Use ultrasound to disperse 0.4 mg of GelMA-PPY solid powder in 96 μL of Gel MA aqueous solution. Add 96 μL of 8.33% PEGDA aqueous solution and 4 μL of 2% polydopamine aqueous solution, and stir to mix until a uniform solution I is obtained.

[0074] Prepare a 0.25% mass volume fraction initiator standard solution: Take 20mL PBS, add it to a brown bottle containing 0.05g initiator LAP, heat it in a 40-50℃ water bath to dissolve for 15 minutes, shake it several times during the period, and set it aside. Take the initiator standard solution to prepare 196μL of 8.24% mass volume fraction GelMA solution, heat it in a 60-70℃ water bath in the dark for 20-30 minutes, shake it several times during the period, and obtain solution II.

[0075] Add solution I to solution II, mix thoroughly, add to the mold, heat in a 50°C water bath under negative pressure to remove bubbles (repeat 3 to 4 times), heat and concentrate in an oven at 30 to 35°C twice, each time for 5 hours, heat and concentrate to a viscous state, avoid excessive drying, and finally use UV405nm light curing to obtain the GelMA-PPY-PDA gel matrix.

[0076] The results are as follows Fig.10 , 11 As shown in Figure 12, the CV curve and impedance curve of the conductive microneedle patch were analyzed by an electrochemical workstation, proving that the hydrogel modified with PPY and PDA has good conductivity. The H-NMR spectrum of GelMA-PPY-PDA has obvious amide groups and hydroxyl groups, indicating that PPY and PDA have been successfully attached to the surface of GelMA.

[0077] Example 4

[0078] Preparation of microneedle patch doped with L-Arg@ZIF-90-TPP nanoparticles

[0079] 3 mg of L-Arg@ZIF-90-TPP nanoparticles were added to the 0.25% photoinitiator solution to disperse and dissolve, and then 30 mg of GelMA-PPY-PDA powder and 126 mg of GelMA powder were added to obtain solution III.

[0080] Solution III was added to solution II of Example 3, and after thorough mixing, the mixture was added to the mold, heated in a 50°C water bath under negative pressure to remove bubbles (repeat 3 to 4 times), and concentrated in an oven at 30-35°C for 2 times, each time for 5 h, and heated and concentrated to a viscous state to avoid excessive drying. Finally, the mixture was cured with UV 405nm light to obtain a microneedle patch doped with L-Arg@ZIF-90-TPP nanoparticles.

[0081] The results are as follows Fig.13 It shows that the microneedle patch has microneedles on its surface, and L-Arg@ZIF-90-TPP nanoparticles are randomly distributed at the needle tips of the NO microneedle patch made of GelMA-PPY-PDA gel matrix.

[0082] Example 5

[0083] 30 mg of L-Arg@ZIF-90-TPP nanoparticles were added to the 0.25% photoinitiator solution to disperse and dissolve. 30 mg of GelMA-PPY-PDA powder and 126 mg of GelMA powder were added to obtain solution IV, which was added to solution II of Example 3. The remaining operations were the same as those of Example 4 to obtain a microneedle patch doped with L-Arg@ZIF-90-TPP nanoparticles.

[0084] The results showed that the needle tips of the prepared NO microneedle patch were randomly distributed, which was in line with the expected preparation effect.

[0085] Example 6

[0086] Experimental study on co-localization of L-Arg@ZIF-90-TPP nanoparticles in neonatal rat cardiomyocytes

[0087] Neonatal rat cardiomyocytes NRCM were extracted and cultured in a culture dish covered with gelatin, with 8000 cells per well. After culturing the cells with cardiomyocyte culture medium for 12 hours, the L-Arg@ZIF-90-TPP nanoparticles prepared in Example 2 were added thereto, and the final content of L-Arg@ZIF-90-TPP nanoparticles was 40 μg / mL. After incubation for 2, 4, 18 and 24 hours, Hoechst dye and mitochondrial dye were added to incubate the cells for 10 minutes, and the cells were washed 3 times with PBS and observed under a laser confocal microscope.

[0088] The results are as follows Fig.14It was shown that there was no obvious green fluorescence in NRCM after incubation for 2h and 4h, indicating that NRCM did not take up a large amount of L-Arg@ZIF-90-TPP nanoparticles. After incubation for 18h and 24h, the red fluorescence of NRCM overlapped with the green fluorescence to a large extent, showing yellow fluorescence, where the green fluorescence came from FITC-labeled L-Arg@ZIF-90-TPP nanoparticles, the red fluorescence indicated mitochondria, and the blue indicated the nucleus. The results showed that L-Arg@ZIF-90-TPP nanoparticles could achieve targeted delivery to mitochondria.

[0089] Example 7

[0090] In vivo fluorescence imaging of microneedle patches doped with L-Arg@ZIF-90-TPP nanoparticles after implantation into the mouse heart

[0091] Construction of myocardial ischemia-reperfusion injury model: 8-week-old C57 male mice were used, instruments were sterilized, animal skin was prepared and disinfected, and mice were fully anesthetized with 1% pentobarbital at a dose of 200 mg / kg intraperitoneally. The mice were placed in a supine position, and then the neck tissue was separated under a microscope and intubated to connect to a ventilator. The respiratory rate was adjusted to 120 times / minute and the tidal volume was 2 mL. A 1.2 cm transverse incision was made between the fourth and fifth intercostal spaces on the left edge of the sternum, and the heart was separated layer by layer to expose the heart. The left anterior descending artery was ligated, and the apex of the heart was whitened due to ischemia. After 30 minutes, the ligature was cut off, the intercostal muscles, chest wall muscles and skin were sutured, and the endotracheal tube was removed after the mouse had reflexes. The postoperative wound was disinfected with iodine, and the mouse was put back into the cage when it could breathe autonomously and crawl on its own. The model was successfully constructed.

[0092] The sham group (sham) was operated in the same manner as above except that the ligature was not performed. The treatment group was immediately affixed with the microneedle patch doped with L-Arg@ZIF-90-TPP nanoparticles prepared in Example 4 after the ligature was removed. The other steps were the same as above. The microneedle patch was attached to the heart with the aid of α-ethyl cyanoacrylate. After 1, 2, and 3 weeks of treatment, the mouse heart was taken and placed under an in vivo fluorescence imager to observe the fluorescence distribution.

[0093] The results are as follows Fig.15 The results showed that the fluorescence signal in the mouse heart persisted for up to 3 weeks, but the signal intensity gradually weakened with time, indicating that the microneedle patch can ensure NO delivery within 3 weeks, and that L-Ar g@ZIF-90-TPP nanoparticles are metabolized out of the body during the treatment process and will not be deposited in the body.

[0094] Example 8

[0095] Experimental study on the improvement of myocardial ischemia-reperfusion injury in mice by the GelMA-PPY-PDA microneedle patch prepared in Example 3 and the GelMA-PPY-PDA microneedle patch doped with L-Arg@ZIF-90-TPP nanoparticles prepared in Example 4

[0096] Eight-week-old C57 male mice were used to establish a mouse myocardial ischemia-reperfusion injury model. The mice with successful modeling were randomly divided into 6 groups, with 10 mice in each group, one group was a negative control group (the mouse myocardial ischemia-reperfusion injury (I / RI) model was not treated), one group was a mouse myocardial ischemia-reperfusion injury model treated with the conductive microneedles prepared in Example 3 (I / RI+GelMA-PPY-PDA group), and one group was a mouse myocardial ischemia-reperfusion injury model treated with the conductive microneedles prepared in Example 4 (I / RI+GelMA-PPY-PDA group). A group of mice were treated with microneedles doped with L-Arg@ZIF-90-TPP nanoparticles to treat myocardial ischemia-reperfusion injury (I / RI+L-Arg@ZIF-90-TPP+GelMA-PPY-PDA group), a group of mice were treated with microneedles doped with L-Arg@ZIF-90-TPP nanoparticles to treat myocardial ischemia-reperfusion injury (I / RI+L-Arg@ZIF-90-TPP+GelMA group), and a group of mice were treated with microneedles doped with L-Arg to treat myocardial ischemia-reperfusion injury (I / RI+L-Arg+GelMA-PPY group). The last group of sham-operated healthy mice served as the positive control group. The heart tissue of the mice was taken to detect myocardial cell apoptosis on the 3rd day of treatment, and the cardiac function of the mice was detected after 3 weeks of treatment.

[0097] The results are as follows Fig.16 and 17 It was shown that after 3 weeks of treatment with the microneedle patch, cardiac ultrasound was used to detect the cardiac function of each group. It was found that the cardiac function of mice in the I / RI group was poor, while the LVEF and LVFS of mice in the I / RI+L-Arg@ZIF-90-TPP+GelM A-PPY-PDA group were significantly improved, indicating that the microneedle patch loaded with L-Arg@ZIF-90-TPP nanoparticles can significantly improve cardiac function and has a good therapeutic effect. The serum of mice after 3 weeks of treatment was taken to detect liver and kidney function indicators. By comparing the positive control group and the negative control group, the conductive microneedles prepared in Example 3 and Example 4 did not cause acute damage to the liver and kidney of mice, indicating that the prepared microneedle patch and ZIF-90 nanocarrier have good biocompatibility and will not cause toxic side effects to normal organs.

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A NO nano-delivery vector targeting myocardial cell mitochondria, characterized in that: The invention comprises a nano-carrier ZIF-90, L-arginine and a surface modification molecule triphenylphosphine.

2. A method for preparing the NO nano-delivery carrier targeting myocardial cell mitochondria according to claim 1, characterized in that: The preparation method comprises the following steps: loading L-arginine on a nanocarrier ZIF-90 by an electrostatic adsorption method to obtain L-Arg@ZIF-90 nanoparticles; and modifying triphenylphosphine molecules on the surface of the L-Arg@ZIF-90 nanoparticles by an EDC / NHS reaction to obtain a NO nano-delivery carrier L-Arg@ZIF-90-TPP targeting myocardial cell mitochondria.

3. The preparation method according to claim 2, characterized in that: The mass ratio of the L-arginine to the nanocarrier ZIF-90 is 1:(0.5-8).

4. The preparation method according to claim 2, characterized in that: The mass ratio of the L-Arg@ZIF-90 nanoparticles, EDC, NHS and triphenylphosphine is 1:(1-2):(0.5-1):(2-3).

5. Use of the NO nano-delivery carrier targeting myocardial cell mitochondria according to claim 1 or the method according to any one of claims 2 to 4 in the preparation of a drug, characterized in that: The drug is any one or both of the following: 1) The drug is a drug for preventing and / or treating myocardial ischemia-reperfusion injury; 2) The drug is a drug that inhibits the production of reactive oxygen species in mitochondria of cells.

6. A NO microneedle patch based on a nano-delivery carrier, characterized in that: The microneedle patch comprises the NO nano delivery carrier targeting myocardial cell mitochondria according to claim 1 and a gel matrix.

7. A method for preparing the NO microneedle patch based on a nano-delivery carrier according to claim 6, characterized in that: The preparation method comprises the following steps: 1) GelMA, PPY and ammonium persulfate are cross-linked to obtain GelMA-PPY; 2) GelMA-PPY, GelMA, PEGDA, and polydopamine were mixed and cross-linked to obtain a GelMA-PPY-PDA gel matrix; 3) L-Arg@ZIF-90-TPP nanoparticles were added to the photoinitiator, and then mixed with the GelMA-PPY-PD A gel matrix, and then photocured to obtain the NO microneedle patch encapsulating the L-Arg@ZIF-90-TPP nanoparticles.

8. The preparation method according to claim 7, characterized in that: The mass ratio of GelMA, PPY and ammonium persulfate in the step 1) is 75:(5-30):(10-50); the mass ratio of GelMA-PPY, GelMA, PEGDA and polydopamine in the step 2) is (4000-6000):(40-60):(80-120):

1.

9. The preparation method according to claim 7, characterized in that: In the step 3), the mass ratio of the GelMA-PPY-PDA gel matrix to the L-Arg@ZIF-90-TPP nanoparticles is (1-10):

1.

10. The NO microneedle patch based on a nano-delivery carrier according to claim 6, or the use of the preparation method according to claim 7 or 8 in the preparation of a pharmaceutical preparation, characterized in that: The pharmaceutical preparation is any one or more of the following pharmaceutical preparations: 1) The pharmaceutical preparation is a pharmaceutical preparation for removing excess ROS in myocardial tissue after myocardial infarction; 2) The pharmaceutical preparation is a pharmaceutical preparation for preventing and / or treating myocardial ischemia-reperfusion injury; 3) The pharmaceutical preparation is a pharmaceutical preparation for preventing and / or treating arrhythmia caused by myocardial ischemia; 4) The pharmaceutical preparation is a pharmaceutical preparation for improving the conduction of electrical signals in myocardial tissue.

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