A system for in situ NO generation in the heart based on bioorthogonal reactions and its applications

Abstract

This invention provides a system for in-situ cardiac NO generation based on bioorthogonal reactions and its application, belonging to the pharmaceutical field. The system of this invention improves the bioavailability of NO in the heart, thereby significantly improving myocardial ischemia-reperfusion injury. Specifically, microneedles are pre-implanted into the heart, and then a delivery system (prepared by modifying L-arginine with trans-cyclooctene to form an L-arginine prodrug, and then preparing a nano-delivery system by electrostatic adsorption of L-Arg-TCO) is injected into the body via intravenous injection. Only when the nano-delivery system circulates to the heart, and the TCO undergoes a bioorthogonal reaction with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine in the microneedles, can the L-arginine prodrug be activated, thereby achieving cardiac-targeted delivery of L-arginine, leading to the enrichment, activation, and conversion of L-arginine into NO in the heart.

Description

Technical Field

[0001] This invention relates to the field of medicine, specifically to a system for in-situ generation of NO in the heart based on bioorthogonal reactions and its applications. Background Technology

[0002] Myocardial infarction is one of the leading causes of cardiovascular death. Timely reperfusion is an effective treatment to prevent myocardial cell necrosis after myocardial infarction. However, blood reperfusion can accelerate and amplify myocardial ischemia-reperfusion injury by inducing myocardial cell death and fibrosis.

[0003] Nitric oxide (NO) is an important gaseous signaling molecule in the cardiovascular system, playing a crucial role in maintaining normal vascular physiological function. NO can reduce the activity of complex I in the mitochondrial electron transport chain by relaxing vascular tone, inhibiting platelet aggregation, and regulating inflammatory responses. This limits the production of reactive oxygen species (ROS) in mitochondria, further preventing the opening of the mitochondrial permeability transition pore (mPTP), reducing cytochrome c release, and inhibiting cardiomyocyte apoptosis. However, the efficacy of NO exhibits a strict dose-dependent effect; excessively high doses can lead to apoptosis and toxicity. Excessive ROS resulting from myocardial ischemia-reperfusion injury can directly react with NO, leading to the formation of the strong oxidant peroxynitrite (ONOO). − The formation of NO and the mediating of the oxidation of non-protein and protein thiols significantly increase the nitration of tyrosine in related proteins in mitochondria, severely impacting cell function and vitality. Furthermore, systemic NO release can lead to side effects such as decreased blood pressure and accelerated heart rate. Therefore, developing precise NO delivery systems to achieve targeted delivery is currently a hot research topic in this field and a bottleneck restricting the clinical application of NO biomaterials. Summary of the Invention

[0004] This invention provides a system for in situ cardiac NO generation based on bioorthogonal reactions and its application. The system of this invention can achieve cardiac-targeted delivery and conversion of L-arginine to NO mediated by bioorthogonal reactions.

[0005] This invention provides a system for in situ cardiac NO generation based on bioorthogonal reactions, including microneedles and a delivery system; The delivery system includes a carrier and an L-arginine prodrug loaded on the carrier; The L-arginine prodrug has the structural formula shown in Formula I: Formula I; The microneedles are grafted with and / or loaded with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine.

[0006] Preferably, the carrier includes ZIF-90.

[0007] Preferably, the mass of the L-arginine prodrug is 38.357 ± 1.7678% of the carrier mass.

[0008] Preferably, the preparation method of the L-arginine prodrug includes the following steps: L-arginine, trans-cyclooctene-succinimide ester, and a first buffer solution were mixed to carry out a first coupling reaction to obtain the L-arginine prodrug.

[0009] Preferably, the molar ratio of L-arginine to trans-cyclooctene-succinimide ester is 1:0.5~10; The temperature of the first coupling reaction is 0~37℃ and the time is 1~48h.

[0010] Preferably, the preparation method of the delivery system includes the following steps: The carrier, L-arginine prodrug, and solvent are mixed and electrostatic adsorption occurs to obtain the delivery system.

[0011] Preferably, the height of the microneedle is 100~300μm; the microneedle is also grafted with an arginase inhibitor; The arginase inhibitor includes L-valine.

[0012] Preferably, the method for preparing the microneedles includes the following steps: Sodium hyaluronate grafted with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine, sodium hyaluronate grafted with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine, photoinitiator and third buffer were mixed and placed in a microneedle mold for heating concentration and photocuring to obtain the microneedles; When the microneedles are also grafted with arginase inhibitors, the mixed raw materials also include acrylated polyethylene glycol NHS ester grafted with arginase inhibitors. The method for preparing the acrylated polyethylene glycol NHS ester grafted with an arginase inhibitor includes the following steps: Arginase inhibitor, acrylated polyethylene glycol NHS ester, and third buffer were mixed and grafted to obtain acrylated polyethylene glycol NHS ester grafted with arginase inhibitor.

[0013] Preferably, the method for preparing the methacrylated sodium hyaluronate grafted with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine includes the following steps: Sodium hyaluronate grafted with methacrylate, NHS, EDC, pH adjuster, (4-(6-methyl-1,2,4,5-tetrazinecyclo-3-yl)phenyl)methylamine hydrochloride, and a second buffer solution were mixed and subjected to a second coupling reaction to obtain the sodium hyaluronate grafted with (4-(6-methyl-1,2,4,5-tetrazinecyclo-3-yl)phenyl)methylamine.

[0014] The present invention also provides the application of the system described above in the preparation of devices to improve myocardial ischemia-reperfusion injury.

[0015] This invention designs a NO in-situ targeted cardiac generation system mediated by bioorthogonal reactions, which improves the bioavailability of NO in the heart and thus significantly improves myocardial ischemia-reperfusion injury. Specifically, microneedles are pre-implanted into the heart, and then the delivery system (prepared by modifying L-arginine with trans-cyclooctene (TCO) to form an L-arginine prodrug (L-Arg-TCO), and then electrostatically adsorbing L-Arg-TCO to form a nano-delivery system) is injected into the body via intravenous injection. Only when the delivery system circulates to the heart and the TCO undergoes a bioorthogonal reaction (IEDDA) with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine in the microneedles can the L-arginine prodrug be activated, thereby achieving targeted cardiac delivery of L-arginine, leading to the enrichment, activation, and conversion of L-arginine into NO in the heart.

[0016] Because microneedle implantation requires open-chest surgery, and in actual clinical treatment, each patient can only undergo open-chest surgery once in their lifetime, the medication in the microneedles cannot be replenished once it is depleted. Even with intravenous injection of L-arginine, it is impossible to control its conversion into NO solely in the heart. This invention utilizes a tail vein injection L-arginine prodrug nanodelivery system, ensuring its conversion into NO solely in the heart. This satisfies both the NO conversion needs in the heart and achieves timely replenishment of the L-arginine prodrug. Therefore, this invention has unique advantages in treating long-term myocardial injury.

[0017] In vivo, L-arginine participates not only in the catalytic reaction of nitric oxide synthase to produce NO, but also in the arginase pathway to convert L-arginine into urea. Especially under ischemia-reperfusion conditions, nitric oxide synthase activity is inhibited, while arginase activity is enhanced, leading to more L-arginine being diverted to the arginase pathway, severely reducing the efficiency of L-arginine conversion to NO. This invention utilizes microneedles to maintain long-term adhesion to the left ventricular myocardium. Furthermore, due to the limited height of the microneedles (100-300 μm), they can only penetrate the myocardial layer, delivering L-valine to the myocardium without entering the heart chambers. This achieves in-situ continuous drug delivery, avoiding the blood circulation of L-valine, thus reducing the side effects of NO treatment on other normal organs. It also achieves synergistic treatment between arginase inhibitors and L-arginine, reduces L-arginine consumption, and improves the efficiency of L-arginine conversion to NO.

[0018] Furthermore, the microneedles (HAMA-Tz microneedles) prepared by the present invention using sodium hyaluronate methacrylate grafted with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine as the raw material can release arginase inhibitors (L-valine) for a long time, thereby inhibiting the arginase pathway from competing for L-arginine and improving the efficiency of L-arginine to NO conversion.

[0019] This invention starts with the local conversion efficiency of nitric oxide in the heart and utilizes bioorthogonal reactions to achieve NO conversion only in the heart and inhibit the arginase pathway. The two work synergistically to improve the bioavailability of NO. Attached Figure Description

[0020] Figure 1 The 1H NMR spectrum of L-Arg-TCO; Figure 2 The image shows the ultraviolet spectrum of L-Arg-TCO. Figure 3 The infrared spectrum of L-Arg-TCO; Figure 4 Figure showing the results of the cytotoxicity experiment of L-Arg-TCO on AC16 cells; Figure 5 TEM image of L-Arg-TCO@ZIF-90 nanoparticles; Figure 6 The particle size distribution of L-Arg-TCO@ZIF-90 is shown. Figure 7 Release the L-Arg-TCO curve for L-Arg-TCO@ZIF-9; Figure 8 Figure showing the results of the cytotoxicity experiment of L-Arg-TCO@ZIF-90 against AC16; Figure 9 The 1H NMR spectrum of HAMA-Tz; Figure 10 The infrared spectrum of HAMA-Tz; Figure 11 The image shows the ultraviolet spectrum of HAMA-Tz. Figure 12 Image of a microneedle; Figure 13 An optical microscope image of a microneedle; Figure 14 A fluorescence microscope for microneedles; Figure 15 The images show the heart fluorescence imaging after microneedle patch transplantation into the heart of a mouse with myocardial ischemia-reperfusion injury and after tail vein injection of L-Arg-TCO@ZIF-90. Figure 16 This is a statistical graph of cardiac fluorescence intensity in in vivo fluorescence imaging. Figure 17 These are images of heart tissue sections after microneedle patch transplantation into the heart of a mouse with myocardial ischemia-reperfusion injury and after tail vein injection of L-Arg-TCO@ZIF-90. Figure 18 Fluorescence images of NO production in endothelial cells from different groups; Figure 19 A statistical graph showing the fluorescence intensity of NO produced by endothelial cells in different groups. Detailed Implementation

[0021] This invention provides a system for in situ cardiac NO generation based on bioorthogonal reactions, including microneedles and a delivery system; The delivery system includes a carrier and an L-arginine prodrug loaded on the carrier; The L-arginine prodrug has the structural formula shown in Formula I: Formula I; The microneedles are grafted with and / or loaded with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine.

[0022] In this invention, the mass of the L-arginine prodrug is 38.357 ± 1.7678% of the carrier mass; the carrier preferably includes ZIF-90. ZIF-90 degrades in response to the mitochondrial environment, thereby releasing the L-arginine prodrug and achieving targeted delivery to myocardial mitochondria.

[0023] In this invention, the preparation method of the L-arginine prodrug preferably includes the following steps: L-arginine, trans-cyclooctene-succinimide ester, and a first buffer solution were mixed to carry out a first coupling reaction to obtain the L-arginine prodrug.

[0024] In this invention, the molar ratio of L-arginine to trans-cyclooctene-succinimide ester is preferably 1:0.5~10. In specific embodiments of this invention, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9 or 1:9.5.

[0025] In this invention, the preferred ratio of L-arginine to the first buffer solution is 2 mg:1 mL; the preferred concentration of the buffer solution is 0.01 mol / L; and the first buffer solution preferably includes PBS buffer.

[0026] The temperature of the first coupling reaction is 0~37℃, and the time is 1~48h. In a specific embodiment of the present invention, the temperature of the first coupling reaction can be 4℃, 8℃, 10℃, 12℃, 15℃, 18℃, 20℃, 25℃, 28℃, 30℃, 32℃ or 35℃, and the time can be 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h or 45h.

[0027] After the first coupling reaction is completed, the present invention preferably further includes: placing the system obtained from the first coupling reaction in a dialysis bag and dialyzing it sequentially in sodium chloride solution and water, and then lyophilizing the dialysis product to obtain the L-arginine prodrug.

[0028] The L-arginine prodrug of this invention can only be activated to form L-arginine and then converted into NO when it encounters a microneedle in the heart and undergoes a bioorthogonal reaction between Tz and TCO.

[0029] In this invention, the preparation method of the delivery system includes the following steps: The carrier, L-arginine prodrug, and solvent are mixed and electrostatic adsorption occurs to obtain the delivery system.

[0030] In this invention, the mixing preferably includes: mixing the carrier with a portion of the solvent to obtain a dispersion; mixing the L-arginine prodrug with the remaining solvent and then mixing it with the dispersion.

[0031] In this invention, the solvent preferably includes one or more of water, methanol, ethanol, and N,N-dimethylformamide.

[0032] In this invention, the mixing is preferably carried out under stirring conditions, the stirring speed is 600 rpm, and the stirring time is preferably 1 hour.

[0033] During the mixing process, the L-arginine prodrug is loaded onto the interior and surface of the carrier through electrostatic adsorption.

[0034] After mixing, the present invention preferably further includes: centrifuging the resulting mixture, and then washing the resulting solid to obtain the delivery system.

[0035] In this invention, the height of the microneedle is preferably 100~300 mm, and in specific embodiments of this invention, it can be 120 μm, 150 μm, 180 μm, 200 μm, 210 μm, 240 μm, 250 μm or 280 μm; the microneedle is preferably also grafted with an arginase inhibitor.

[0036] In this invention, the method for preparing the microneedles includes the following steps: Sodium hyaluronate grafted with (4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)methylamine, sodium hyaluronate grafted with (4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)methylamine, photoinitiator and third buffer solution were mixed and placed in a microneedle mold for heating concentration and photocuring to obtain the microneedles.

[0037] In this invention, the mass ratio of sodium methacrylated hyaluronic acid to sodium methacrylated hyaluronic acid grafted with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine is preferably 9:0.1 to 5. In specific embodiments of this invention, it can be 9:0.2, 9:0.5, 9:0.8, 9:1, 9:2, 9:3 or 9:4.

[0038] In this invention, the method for preparing sodium methacrylate grafted with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine preferably includes the following steps: Sodium hyaluronate grafted with methacrylate, NHS, EDC, pH adjuster, (4-(6-methyl-1,2,4,5-tetrazinecyclo-3-yl)phenyl)methylamine hydrochloride, and a second buffer solution were mixed and subjected to a second coupling reaction to obtain the sodium hyaluronate grafted with (4-(6-methyl-1,2,4,5-tetrazinecyclo-3-yl)phenyl)methylamine.

[0039] In this invention, the pH value of the mixture obtained by mixing is preferably 6; the pH adjuster preferably includes hydrochloric acid; and the concentration of the hydrochloric acid is preferably 0.1M.

[0040] In this invention, the mixing preferably includes mixing sodium methacrylated hyaluronic acid and the second buffer solution, and then mixing them with the first pH adjuster to obtain a first mixture; The first mixture is mixed with NHS, EDC, and a second pH adjuster to obtain a second mixture; The second mixture was mixed with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine hydrochloride and the remaining pH adjuster.

[0041] In this invention, the preferred ratio of sodium methacrylated hyaluronic acid to the second buffer solution is 40 mg: 1 mL; the second buffer solution preferably includes MES buffer solution; and the preferred concentration of the second buffer solution is 0.1 M.

[0042] In this invention, the pH value of the first mixture is preferably 5.5.

[0043] In this invention, the mass ratio of sodium methacrylated hyaluronic acid to NHS is preferably 2:1 to 3, and in specific embodiments of this invention, it can be 2:1.2, 2:1.5, 2:1.8, 2:2, 2:2.2, 2:2.5 or 2:2.8; the molar ratio of NHS to EDC is preferably 1 to 1.2:1.

[0044] In this invention, the pH value of the second mixture is preferably 5.5.

[0045] In this invention, the preferred mass ratio of sodium methacrylated hyaluronic acid to (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine hydrochloride is 2:1 to 3. In specific embodiments of this invention, it can be 2:1.2, 2:1.5, 2:1.8, 2:2, 2:2.2, 2:2.5, or 2:2.8.

[0046] In this invention, the temperature of the second coupling reaction is preferably room temperature, and the time is preferably 24 hours. In the second coupling reaction, the COOH on the surface of HAMA reacts with the NH2 of (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine hydrochloride (Tz-Me-amine).

[0047] After the second coupling reaction is completed, the present invention preferably further includes: placing the system obtained from the second coupling reaction in a dialysis bag and dialyzing it sequentially in sodium chloride solution and water, and then freeze-drying the dialysis product to obtain the methacrylated sodium hyaluronate grafted with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine.

[0048] When the microneedles are also grafted with an arginase inhibitor, the raw materials for mixing also include acrylated polyethylene glycol NHS ester grafted with an arginase inhibitor. Preferably, a photoinitiator is added during the preparation of the acrylated polyethylene glycol NHS ester grafted with an arginase inhibitor.

[0049] In this invention, the mass ratio of the total mass of the sodium methacrylated hyaluronic acid and the sodium methacrylated hyaluronic acid grafted with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine to the mass of the photoinitiator is preferably 60:0.1 to 1. In specific embodiments of this invention, it can be 60:0.1, 60:0.2, 60:0.3, 60:0.4, 60:0.5, 60:0.6, 60:0.7, 60:0.8, or 60:0.9. The initiator preferably includes lithium phenyl(2,4,6-trimethylbenzoyl)phosphate.

[0050] The preferred method for preparing the acrylated polyethylene glycol NHS ester grafted with an arginase inhibitor includes the following steps: L-valine, acrylated polyethylene glycol NHS ester and third buffer were mixed and grafted to obtain acrylated polyethylene glycol NHS ester grafted with arginase inhibitor. More preferably, the method includes the following steps: mixing L-valine, acrylamide-modified polyethylene glycol NHS ester, photoinitiator and third buffer solution to carry out a grafting reaction to obtain acrylamide-modified polyethylene glycol NHS ester grafted with arginase inhibitor.

[0051] In this invention, the mixing preferably includes mixing the photoinitiator with a third buffer solution to obtain a photoinitiator solution; and then mixing the photoinitiator solution sequentially with L-valine and acrylated polyethylene glycol NHS ester. Dissolving the raw material for preparing acrylated polyethylene glycol NHS ester grafted with an arginase inhibitor in the photoinitiator solution can reduce the amount of water used.

[0052] In this invention, the preferred mass ratio of L-valine to acrylamide polyethylene glycol NHS ester is 1 to 35:1. In specific embodiments of this invention, it can be 1:2, 5:1, 8:1, 10:1, 12:1, 15:1, 18:1, 20:1, 22:1, 25:1, 28:1, 30:1, or 32:1.

[0053] In this invention, the preferred mass ratio of L-valine to sodium methacrylated hyaluronic acid is 2:45.

[0054] In this invention, the preferred ratio of L-valine to photoinitiator solution is 2 mg: 1 mL; the third buffer solution preferably includes PBS buffer; and the pH of the PBS buffer solution is preferably 7.4.

[0055] In this invention, after mixing and before grafting reaction, the mixture obtained by mixing is further subjected to membrane filtration for sterilization.

[0056] In this invention, the grafting reaction temperature is preferably 4°C, and the time is preferably 18~24h. In specific embodiments of this invention, the grafting reaction time can be 19h, 20h, 21h, 22h or 23h.

[0057] In this invention, the grafting reaction is carried out under light-protected conditions.

[0058] The NHS group on acryloylpolyethylene glycol NHS ester undergoes an EDC / NHS reaction with L-valine, achieving the grafting of L-valine.

[0059] The present invention preferably further includes vacuum degassing after the material is placed in the mold and before heating and concentration.

[0060] In this invention, the preferred temperature for heating and concentration is 30-35°C, the preferred time is 4-6 hours, and the preferred number of heating and concentration cycles is 3-4.

[0061] In this invention, the wavelength of the light used for photocuring is preferably 405 nm, and the light intensity is preferably 30 mW / cm². 2 .

[0062] The present invention also provides the application of the system described above in the preparation of devices to improve myocardial ischemia-reperfusion injury.

[0063] The following detailed description, in conjunction with embodiments, illustrates the system for in situ cardiac NO generation based on bioorthogonal reactions provided by this invention and its applications. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0064] In this example, the microneedle has a length of 200 micrometers.

[0065] Example 1 Preparation of L-Arg-TCO 10 mg of L-arginine and TCO-NHS ester were dissolved in 5 mL of 0.01 mol / L PBS solution at a molar ratio of 1:1.5 and the pH was adjusted to 7.2-8.5 with NaOH. The reaction was carried out overnight in the dark at 4°C. The dialysis bag (200 Da) was dialyzed in 1% NaCl aqueous solution and then dialyzed in deionized water. The product was collected, lyophilized, and stored at 4°C.

[0066] Figure 1 The 1H NMR spectrum of L-Arg-TCO.

[0067] Figure 2 This is the ultraviolet spectrum of L-Arg-TCO.

[0068] Figure 3The image shows the infrared spectrum of L-Arg-TCO.

[0069] Figure 4 The figure shows the results of the cytotoxicity experiment of L-Arg-TCO on AC16 cells.

[0070] Figures 1-3 The result shows that L-Arg-TCO has been successfully synthesized.

[0071] Table 1. Results of L-Arg-TCO cytotoxicity assays on AC16 cells.

[0072] The MTT assay confirmed that L-Arg-TCO did not significantly inhibit the survival rate of AC16 cells, indicating that L-Arg-TCO has low cardiocytogenicity.

[0073] Preparation of L-Arg-TCO@ZIF-90 nanoparticles ①L-Arg-TCO was dissolved in ultrapure water at a concentration of 10 mg / mL; ②ZIF-90 was dissolved in ultrapure water by sonication for 15 min to achieve a concentration of 10 mg / mL; ③ Take the corresponding volume of L-Arg-TCO and ZIF-90 according to the ZIF-90:L-Arg-TCO mass ratio of 2:2, mix them, react at 600 rpm for 1 h, centrifuge at 10000 rpm for 10 min, discard the supernatant, and wash twice to obtain the final product.

[0074] Figure 5 TEM image of L-Arg-TCO@ZIF-90 nanoparticles.

[0075] Figure 6 This is the particle size distribution diagram of L-Arg-TCO@ZIF-90.

[0076] Depend on Figures 5-6 It is known that the particle size of L-Arg-TCO@ZIF-90 nanoparticles is around 100 nm.

[0077] Figure 7 The curve of L-Arg-TCO release for L-Arg-TCO@ZIF-9 is shown. The experimental method is described in "Conductive Microneedle Patch with Mitochondria-localized Generation of Nitric Oxide Promotes Heart Repair after Ischemia-Reperfusion Therapy".

[0078] Depend on Figure 7 It is known that nanoparticles can release L-Arg-TCO in acidic and ATP-containing solutions.

[0079] Figure 8 The figure shows the results of the cytotoxicity experiment of L-Arg-TCO@ZIF-90 against AC16. The incubation time was 24 h, n=6. The experimental method is described in "Conductive Microneedle Patch with Mitochondria-localized Generation of Nitric Oxide Promotes Heart Repair after Ischemia-Reperfusion Therapy".

[0080] Depend on Figure 8 It can be seen that the survival rate of AC16 cells remains high.

[0081] Preparation of HAMA-Tz ① Dissolve 200 mg of HAMA in 5 mL of 0.1 M MES solution (it was personally tested that it can be completely dissolved by magnetic stirring at room temperature for about 40 min) to obtain a 40 g / L solution, and then adjust the pH to 5.5 with 0.1 M hydrochloric acid.

[0082] ② Add 165.6 mg of EDC to ① and stir for 20 min, then add 122.8 mg of NHS. Adjust the pH to 5.5 with 0.1 M hydrochloric acid and stir at room temperature for 30 min. Add 175.3 mg of Tz-Me-amine and adjust the pH to 6.0. Stir at room temperature for 24 h, then dialyze in 1% NaCl aqueous solution using a 12 kDa dialysis bag (dialysis bag 12 kDa), and then dialyze in deionized water (dialysis bag 12 kDa). Then freeze-dry the product to obtain HAMA-Tz powder and store at 4 °C.

[0083] Figure 9 The 1H NMR spectrum is HAMA-Tz.

[0084] Figure 10 The image shows the infrared spectrum of HAMA-Tz.

[0085] Figure 11 This is the ultraviolet spectrum of HAMA-Tz.

[0086] Figures 9-11 This indicates that HAMA-Tz possesses the characteristic chemical structures of both HAMA and Tz, demonstrating that HAMA-Tz was successfully synthesized.

[0087] Preparation of HAMA-Tz microneedle patches Dissolve 90 mg HAMA and 30 mg HAMA-Tz in 2 mL of 0.5% mg / mL lithium phenyl (2,4,6-trimethylbenzoyl) phosphate solution to prepare a mixed hydrogel solution of HAMA and HAMA-Tz. Spread the prepared mixed hydrogel solution of HAMA and HAMA-Tz evenly on top to form a microneedle patch. Defoaming was performed by heating in a 50℃ water bath under negative pressure (repeated 4 times). The mixture was then concentrated twice in an oven at 30-35℃ (each time for 4-6 hours) until it reached a viscous state. Avoid excessive drying. Finally, the mixture was cured with UV 405nm light to obtain the HAMA-Tz hydrogel.

[0088] Figure 12 This is a picture of a microneedle.

[0089] Figure 13 This is an optical microscope image of a microneedle.

[0090] Figure 13 An optical microscope image of a microneedle; Figure 14 This is a fluorescence microscope for microneedles.

[0091] A myocardial ischemia-reperfusion injury model was established using 8-week-old C57 male mice. After microneedles were implanted into the heart of the mouse with myocardial ischemia-reperfusion injury and the L-arginine prodrug nanodelivery system was injected into the tail vein, the in vivo fluorescence imaging showed the heart fluorescence image.

[0092] Figure 15 Fluorescence imaging of the heart after HAMA-Tz microneedle patch transplantation into the heart of a mouse with myocardial ischemia-reperfusion injury and after tail vein injection of L-Arg-TCO@ZIF-90. Figure 15 The "Merge" image represents two colors of fluorescence superimposed on each other, while "HAMA-Tz" represents a mouse heart implanted with a microneedle of HAMA-Tz.

[0093] Figure 15 The results showed that the fluorescence signal of the microneedles was present in mouse heart tissue, but the fluorescence signal of the L-arginine nanodelivery system gradually accumulated in mouse heart tissue, indicating that bioorthogonal reactions can promote the accumulation of L-arginine prodrug in heart tissue.

[0094] Figure 16 This is a statistical graph of fluorescence intensity in in vivo fluorescence imaging.

[0095] Depend on Figure 16 The fluorescence intensity after 1 hour is: 1.2508 ± 0.46983 (×10⁻⁶) 8 The fluorescence intensity at 4 h was: 8.69867 ± 0.55864 (×10⁻⁶). 8The fluorescence intensity at 8 h was: 12.297 ± 3.73697 (×10⁻⁶). 8 The fluorescence intensity at 16 h was 9.7 ± 2.65712 (×10⁻⁶). 8 At 8 hours, the fluorescence intensity in the heart was the strongest, indicating that a large number of L-Arg-TCO@ZIF-90 nanoparticles accumulated in the heart 8 hours after injection.

[0096] Microneedle patches were implanted into the hearts of mice with myocardial ischemia-reperfusion injury. Eight hours after tail vein injection of L-Arg-TCO@ZIF-90, the mice were sacrificed, and the hearts were removed, frozen sections were prepared, and nuclear staining was performed using DAPI. Fluorescence distribution was observed under a fluorescence microscope. Results... Figure 17 As shown in the figure. In the slice, the green fluorescence represents L-Arg-TCO@ZIF-90 nanoparticles, and the red fluorescence represents HAMA-Tz hydrogel. The distribution of red and green fluorescence can be observed at the molecular level.

[0097] Figure 17 These are tissue sections of a mouse heart after microneedle patch transplantation into the heart of a mouse with myocardial ischemia-reperfusion injury and after tail vein injection of L-Arg-TCO@ZIF-90.

[0098] Figure 17 The results showed that the red fluorescence representing microneedles in the heart tissue overlapped with the green fluorescence representing L-arginine, indicating that it could enrich the L-arginine prodrug nanodelivery system in the heart under the mediation of a bioorthogonal reaction. L-valine-loaded hydrogel 1) Weigh 4 mg of L-valine (L-Nva) and dissolve it in 2 mL of PBS solution (pH 7.4) containing 0.5 mg / mL of the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphorylated (LAP). Then, add 135.58 mg of AC-PEG-NHS (acryloylated polyethylene glycol NHS ester) to the above solution and vortex for 5 min under light-protected conditions. After sterilization by filtration, the resulting mixture is incubated at 4°C in the dark for 18 h to allow AC-PEG-NHS and L-valine to react fully, generating an AC-PEG-valine (AC-PEG-Nva) conjugate.

[0099] 2) Take 90 mg HAMA (methacrylamide hyaluronic acid) and 30 mg HAMA-Tz (tetraazine-modified methacrylamide hyaluronic acid), dissolve them together in the solution obtained in 1) by fully reacting AC-PEG-NHS with L-valine, and prepare a HAMA / HAMA-Tz+LNva mixed hydrogel precursor solution and then sterilize it.

[0100] Using no precursor solution as a control group, 100 μL of HAMA / HAMA-Tz+LNva mixed hydrogel precursor solution, the aforementioned HAMA and HAMA-Tz mixed hydrogel solution, or HAMA hydrogel precursor solution (prepared by dissolving 120 mg HAMA in 2 mL of 0.5% mg / mL lithium phenyl (2,4,6-trimethylbenzoyl) phosphate solution) was slowly added to a 48-well plate in a clean bench to ensure uniform spreading of the liquid. The plate was then irradiated with a 405 nm UV light source to solidify it, and the uncured precursor solution in the wells was aspirated and discarded. Add an appropriate amount of serum-containing culture medium to each well to soak the hydrogel overnight, then discard the culture medium and add an endothelial cell suspension of appropriate density. Place the culture plate in an incubator for culture. The cell suspension density at the time of cell plating is 1,000,000 / mL. Add 200 μL to each well. After the cells have completely adhered to the plate, add L-Arg-TCO@ZIF-90 and incubate for 24 hours to detect NO.

[0101] Figure 18 Fluorescence images of NO production in endothelial cells from different groups.

[0102] Depend on Figure 18 It is known that the hydrogel loaded with L-valine inhibits arginine activity by releasing L-valine, thus producing the most NO in endothelial cells with the same L-Arg-TCO@ZIF-90 concentration.

[0103] Figure 19 Statistical graph of NO fluorescence intensity produced by endothelial cells in different groups: Control: 1.0000±0.205634 times, HAMA: 1.211317±0.166441 times, HAMA-Tz: 2.741312±0.521462 times, HAMA-Tz+L-Nva: 5.244971±0.596055 times.

[0104] Figures 18-19 The results showed that L-valine released by the microneedles could significantly inhibit the activity of arginase, thereby increasing NO production.

[0105] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A system for in-situ generation of NO in the heart based on bioorthogonal reactions, characterized in that, Including microneedles and delivery systems; The delivery system includes a carrier and an L-arginine prodrug loaded on the carrier; The L-arginine prodrug has the structural formula shown in Formula I: Equation I; The microneedles are grafted with and / or loaded with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine.

2. The system according to claim 1, characterized in that, The carrier includes ZIF-90.

3. The system according to claim 1 or 2, characterized in that, The mass of the L-arginine prodrug is 38.357 ± 1.7678% of the carrier mass.

4. The system according to claim 1, characterized in that, The preparation method of the L-arginine prodrug includes the following steps: L-arginine, trans-cyclooctene-succinimide ester, and a first buffer solution were mixed to carry out a first coupling reaction to obtain the L-arginine prodrug.

5. The system according to claim 4, characterized in that, The molar ratio of L-arginine to trans-cyclooctene-succinimide ester is 1:0.5~10; The temperature of the first coupling reaction is 0~37℃ and the time is 1~48h.

6. The system according to claim 1, characterized in that, The preparation method of the delivery system includes the following steps: The carrier, L-arginine prodrug, and solvent are mixed and electrostatic adsorption occurs to obtain the delivery system.

7. The system according to claim 1, characterized in that, The height of the microneedle is 100~300μm; the microneedle is also grafted with an arginase inhibitor; The arginase inhibitor includes L-valine.

8. The system according to claim 7, characterized in that, The preparation method of the microneedles includes the following steps: Sodium hyaluronate grafted with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine, sodium hyaluronate grafted with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine, photoinitiator and third buffer were mixed and placed in a microneedle mold for heating concentration and photocuring to obtain the microneedles; When the microneedles are also grafted with arginase inhibitors, the mixed raw materials also include acrylated polyethylene glycol NHS ester grafted with arginase inhibitors. The method for preparing the acrylated polyethylene glycol NHS ester grafted with an arginase inhibitor includes the following steps: Arginase inhibitor, acrylated polyethylene glycol NHS ester, and third buffer were mixed and grafted to obtain acrylated polyethylene glycol NHS ester grafted with arginase inhibitor.

9. The system according to claim 8, characterized in that, The method for preparing sodium methacrylate grafted with (4-(6-methyl-1,2,4,5-tetraazinecyclo-3-yl)phenyl)methylamine includes the following steps: Sodium hyaluronate grafted with methacrylate, NHS, EDC, pH adjuster, (4-(6-methyl-1,2,4,5-tetrazinecyclo-3-yl)phenyl)methylamine hydrochloride, and a second buffer solution were mixed and subjected to a second coupling reaction to obtain the sodium hyaluronate grafted with (4-(6-methyl-1,2,4,5-tetrazinecyclo-3-yl)phenyl)methylamine.

10. The use of the system according to any one of claims 1 to 9 in the preparation of devices to improve myocardial ischemia-reperfusion injury.

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