A synthetic method for a nano-delivery system that mediates the reactive nitrogen / reactive oxygen species-sensitized "ferroptosis" effect through NO gas signaling molecules

Abstract

A method for synthesizing a nano-delivery system for a NO gas signaling molecule-mediated reactive nitrogen / reactive oxygen sensitization "ferroptosis" effect, which relates to a method for synthesizing a NO gas signaling molecule nano-delivery system. The purpose of the present invention is to solve the problems that existing NO donors or releasing molecules are insufficiently representative, and NO diffuses freely and rapidly in the body, has a short biological lifespan, lacks effective accumulation in tumor sites, and has low bioavailability. Methods: 1. Synthesis of ZIF@MnO2 nanoenzyme; 2. Synthesis of ZIF@MnO2-NO nano-delivery system; 3. Synthesis of PEG / ZIF@MnO2-NO nano-delivery system. The present invention proposes a controllable metal-organic framework (MOFs)-modified biodegradable nano-enzyme for ROS / RNS-mediated "ferroptosis". The present invention can obtain a nano-delivery system for a NO gas signaling molecule-mediated reactive nitrogen / reactive oxygen sensitization "ferroptosis" effect.

Description

Technical Field

[0001] The invention relates to a method for synthesizing a NO gas signal molecule nano-delivery system. Background Art

[0002] Triple-negative breast cancer (TNBC) is a severely invasive and highly proliferative subtype of breast cancer, and it is still the most challenging subtype of breast cancer to treat. TNBC has a rapid recurrence and poor prognosis, and there is a lack of effective treatments in clinical practice. Multi-omics analysis showed that "ferroptosis"-related pathways are highly enriched in TNBC subtypes. Among them, TNBC is highly sensitive to glutathione peroxidase (GPX4) inhibitors. Therefore, GPX4 may be a metabolic therapeutic target for TNBC tumors. Studies have found that nitric oxide (NO) can cause tumor cells to die due to energy metabolism disorders by affecting cellular energy metabolism, and can also be inhibited by System Xc － The pathway cooperates with GPX4 to inhibit the depletion of reduced glutathione (GSH) in the tumor microenvironment, specifically sensitizing the "ferroptosis" effect.

[0003] NO was the first gaseous molecule discovered to participate in cell signaling. It plays a crucial role in a variety of physiological responses, including cardiovascular regulation, immune regulation, and neurotransmission, in ways that are either dependent or independent of cyclic guanosine monophosphate. NO is a small, fat-soluble gas molecule produced in the human body from L-arginine by three different nitric oxide synthases (NOS): neuronal (nNOS / NOS1), inducible (iNOS / NOS2), and endothelial (eNOS / NOS3). NO can permeate cell membranes and interact with other molecules to directly participate in free radical processes (ROS) or activate free radicals (RNS). However, current research lacks representative NO donors or releasing molecules, and NO diffuses rapidly in vivo, has a short biological lifespan, lacks effective accumulation in tumors, and has low bioavailability. Therefore, research on the targeted enrichment, controlled release, and release patterns of NO in vivo is crucial.

[0004] Studies have shown that RNS species are highly related to the process of ferroptosis. RNS is a series of free radicals and nitrogen-containing compounds with high oxidative activity derived from the interaction between NO and ROS free radicals in the body

[19] . The RNS molecular family is centered on NO and includes peroxynitrite anions (ONOO － ), nitrite ion (NO2 － ), nitroxyl anion (NO － ), nitrogen dioxide (NO2) and nitrogen trioxide (N2O3), etc. Among them, ONOO －Produced by the reaction of NO and superoxide, ONOO is a more stable and active oxidant and nitrating agent than NO, and is also the main component causing nitrification stress. Similar to the physiological effects mediated by NO, ONOO － It can participate in the signal transduction process as a signal molecule, react with most biological molecules such as proteins, lipids and nucleic acids, promote the oxidation or nitration of biological molecules and cause cell damage.

[0005] Therefore, in response to the bottleneck in TNBC treatment and based on the feasibility of NO gas-sensitized "ferroptosis" treatment, it is of great significance to design a nano-delivery system in which NO gas signaling molecules mediate the reactive nitrogen-sensitized "ferroptosis" effect to achieve the "on-demand" release of exogenous NO gas in TNBC. Summary of the Invention

[0006] The purpose of the present invention is to solve the problems of insufficient representation of existing NO donors or releasing molecules, rapid free diffusion of NO in the body, short biological lifespan, lack of effective accumulation in tumor sites, and low bioavailability, and to provide a method for synthesizing a nano-delivery system that mediates the reactive nitrogen / reactive oxygen sensitization "ferroptosis" effect of NO gas signaling molecules.

[0007] In the present invention, a manganese oxide (MnO2) shell is in situ grown on the hollow MOFs skeleton by potassium permanganate etching, and an exogenous NO donor is loaded into the multilayer mesoporous structure of the hollow MOFs to construct a tumor microenvironment-responsive NO gas signaling molecule nano-delivery system.

[0008] The present invention provides a method for synthesizing a nano-delivery system for the NO gas signaling molecule-mediated reactive nitrogen / reactive oxygen species-sensitized "ferroptosis" effect, which is specifically accomplished by the following steps:

[0009] 1. Synthesis of ZIF@MnO2 nanozymes:

[0010] First, the metal organic framework material was dissolved in water, then stirred in an ice bath, KMnO4 solution was added, and stirring was continued in an ice bath, and finally centrifuged to obtain ZIF@MnO2 nanozyme;

[0011] The metal organic framework material described in step 1 is ZIF-8, ZIF-67 or ZIF-90;

[0012] 2. Synthesis of ZIF@MnO2-NO Nanodelivery System:

[0013] First, the NO donor was dissolved in water, and then the pH value of the system was adjusted to 7.0. Then, the ZIF@MnO2 nanozyme was added, stirred in the dark, centrifuged, and the solid was collected and washed to obtain the ZIF@MnO2-NO nanodelivery system.

[0014] 3. Synthesis of PEG / ZIF@MnO2-NO Nanodelivery System:

[0015] First, the ZIF@MnO2-NO nanodelivery system was mixed with polyethylene glycol under ultrasound, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added. The reaction was stirred and finally centrifuged and the solid was collected to obtain the PEG / ZIF@MnO2-NO nanodelivery system, which is a nanodelivery system for the NO gas signal molecule-mediated reactive nitrogen / reactive oxygen sensitization "ferroptosis" effect.

[0016] Beneficial effects of the present invention:

[0017] This paper proposes a regulatable metal-organic framework (MOFs) modified biodegradable nanozyme for ROS / RNS-mediated ferroptosis, in which a two-step depletion and inhibition strategy of GSH can simultaneously activate and amplify the ferroptosis process. The constructed MOFs nanozyme has redox enzyme-like activities, including peroxidase, superoxide oxidase and catalase mimicking activities, which can generate ·OH and ·O2 respectively after initial activation. - and O2 to enhance oxidative stress and alleviate hypoxic conditions. The NO released by the exogenous donor can then react with O2 - The reaction produces highly toxic ONOO - , enhancing nitrification stress and ultimately achieving the ROS / RNS-mediated "ferroptosis" process. Since GSH is an important intracellular antioxidant that can reduce oxidative damage, the present invention proposes a two-step strategy for scavenging GSH. On the one hand, MOFs-modified nanozymes with GSH scavenging ability can inhibit GPX4 enzyme activity, thereby increasing ROS accumulation; on the other hand, the generated RNS can downregulate SystemXc - It inhibits SLC7A11 and glutathione reductase in the mitochondria, inactivating the cystine / cysteine ​​redox cycle, thereby limiting the supply of intracellular cysteine ​​and inhibiting GSH regeneration.

[0018] The present invention can obtain a nano delivery system for the "ferroptosis" effect sensitized by NO gas signal molecules and activated nitrogen / oxygen. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 TEM images of ZIF-8, ZIF-67, and ZIF-90, where a is ZIF-8 prepared in Example 2, b is ZIF-67 prepared in Example 1, and c is ZIF-90 prepared in Example 3;

[0020] Figure 2TEM, HAADF-STEM and element scanning images, in which a is the potassium permanganate oxidation mechanism diagram, b is the TEM image of ZIF-8@MnO2 solid before etching, c is the TEM image of ZIF-8@MnO2 hollow after etching, d is the HAADF-STEM and element scanning image of ZIF-8@MnO2, e is the TEM image of ZIF-67@MnO2 solid before etching, f is the TEM image of ZIF-67@MnO2 hollow after etching, g is the HAADF-STEM and element scanning image of ZIF-67@MnO2, h is the TEM image of ZIF-90@MnO2 solid before etching, i is the TEM image of ZIF-90@MnO2 hollow after etching, and j is the HAADF-STEM and element scanning image of ZIF-90@MnO2;

[0021] Figure 3 XRD patterns, in which (a) is the XRD pattern of ZIF-8, ZIF-8@MnO2 solid before etching (ZIF-8-S@MnO2) and ZIF-8@MnO2 hollow after etching (ZIF-8-H@MnO2), (b) is the XRD pattern of ZIF-67, ZIF-67@MnO2 solid before etching (ZIF-67@MnO2) and ZIF-67@MnO2 hollow after etching (ZIF-67@MnO2), and (c) is the XRD pattern of ZIF-90, ZIF-90@MnO2 solid before etching and ZIF-90@MnO2 hollow after etching;

[0022] Figure 4 The full XPS spectrum of PEG / ZIF-67@MnO2-LA and the high-resolution XPS spectra of Mn 2p and Co 2p;

[0023] Figure 5 N2 adsorption-desorption curves of ZIF-67 and ZIF-67@MnO2, where 1 is ZIF-67 and 2 is ZIF-67@MnO2;

[0024] Figure 6 Zeta potential diagrams of ZIF-67, ZIF-67@MnO2, ZIF-67@MnO2-LA and PEG / ZIF-67@MnO2-LA;

[0025] Figure 7 pH and GSH response decomposition curve of PEG / ZIF-67@MnO2-LA;

[0026] Figure 8Figure 2 is the degradation transmission photo of PEG / ZIF-67@MnO2-LA under different GSH conditions and the percentage of GSH consumed. Figure a is the degradation transmission photo, b is the mechanism diagram of DTNB detection of GSH, and c is the percentage of GSH consumed by different concentrations of PEG / ZIF-67@MnO2-LA.

[0027] Figure 9 To explore the CAT enzyme activity of PEG / ZIF-67@MnO2-LA, Figure a is the dissolved oxygen curve of different solutions, and b is the Michaelis-Menten fitting curve under different conditions;

[0028] Figure 10 To explore the generation of ·OH in PEG / ZIF-67@MnO2-LA;

[0029] Figure 11 To investigate the OXD enzyme activity of PEG / ZIF-67@MnO2-LA;

[0030] Figure 12 To investigate the NO release performance of PEG / ZIF-67@MnO2-LA;

[0031] Figure 13 To explore the therapeutic properties of PEG / ZIF-67@MnO2-LA at the cellular level. DETAILED DESCRIPTION

[0032] Specific embodiment 1: This embodiment provides a method for synthesizing a nano-delivery system for the NO gas signaling molecule-mediated reactive nitrogen / reactive oxygen sensitization "ferroptosis" effect, which is specifically completed by the following steps:

[0033] 1. Synthesis of ZIF@MnO2 nanozymes:

[0034] First, the metal organic framework material was dissolved in water, then stirred in an ice bath, KMnO4 solution was added, and stirring was continued in an ice bath, and finally centrifuged to obtain ZIF@MnO2 nanozyme;

[0035] The metal organic framework material described in step 1 is ZIF-8, ZIF-67 or ZIF-90;

[0036] 2. Synthesis of ZIF@MnO2-NO Nanodelivery System:

[0037] First, the NO donor was dissolved in water, and then the pH value of the system was adjusted to 7.0. Then, the ZIF@MnO2 nanozyme was added, stirred in the dark, centrifuged, and the solid was collected and washed to obtain the ZIF@MnO2-NO nanodelivery system.

[0038] 3. Synthesis of PEG / ZIF@MnO2-NO Nanodelivery System:

[0039] First, the ZIF@MnO2-NO nanodelivery system was mixed with polyethylene glycol under ultrasound, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added. The reaction was stirred and finally centrifuged and the solid was collected to obtain the PEG / ZIF@MnO2-NO nanodelivery system, which is a nanodelivery system for the NO gas signal molecule-mediated reactive nitrogen / reactive oxygen sensitization "ferroptosis" effect.

[0040] MOFs are a series of crystalline porous materials coordinated by a metal-containing core (metal ions and clusters) and an organic linker (negatively charged ligand). Due to the tunability of their composition and structure, MOFs-engineered nanozymes have attracted widespread attention due to their various framework structures, high specific surface area, adjustable crystal size, multi-enzyme activity and good biocompatibility. Here, zeolitic imidazolate frameworks (ZIFs), which combine the advantages of molecular sieves and MOFs, were rationally constructed as multifunctional nanozymes for delivering NO gas signaling molecules for ROS / RNS-mediated "ferroptosis". Under neutral conditions, by reducing KMnO4, the MnO2 shell grows directly on the outer layer, thereby amplifying the catalytic effect and forming a hollow mesoporous structure for further loading the NO donor L-arginine. The MOFs nanozyme has redox enzyme-like activity, including peroxidase, oxidase and catalase mimetic activity, and can be activated: 1) to generate ·OH for ROS-mediated "ferroptosis"; 2) to generate superoxide·O2 - , used to react with NO, thereby achieving RNS-mediated "ferroptosis"; 3) Produce O2 to alleviate hypoxic conditions. Then, under the catalysis of iNOS, L-arginine releases NO and O2 - The reaction produces toxic ONOO - , enhancing nitrification stress, and ultimately achieving effective "ferroptosis" mediated by synergistic ROS / RNS. This embodiment has a stable process, a mature method, and a reaction system that is environmentally friendly. The proposed process route can successfully synthesize a gas signaling molecule delivery system and has good application prospects. This embodiment discloses a method for synthesizing a nano-delivery system for the "ferroptosis" effect mediated by NO gas signaling molecules and sensitized by reactive nitrogen / reactive oxygen species, which has good application prospects and pioneering innovation guidance.

[0041] The nano-delivery system of NO gas signaling molecules mediated reactive nitrogen / reactive oxygen sensitization "ferroptosis" effect prepared in this embodiment is applied to the field of gas signaling molecule-mediated tumor treatment, and specifically sensitizes the "ferroptosis" effect by inducing oxidative / nitrogenic stress through NO. In addition, for the reducing substance GSH in the tumor microenvironment, the nano-delivery system with GSH scavenging ability can inactivate the activity of GPX4, thereby increasing the accumulation of ROS / RNS and enhancing the susceptibility of tumor cells to "ferroptosis". The generated RNS can also downregulate SystemXc - The SLC7A11 and glutathione reductase activity in the cell inactivate the cystine / cysteine ​​redox cycle, thereby limiting the supply of cysteine ​​in the cell and inhibiting the resurrection of GSH. The two-step GSH inhibition strategy largely breaks the imbalance of the intracellular oxidation and antioxidant systems, and maximizes the therapeutic effect of "ferroptosis". Therefore, the biodegradable MOFs engineered gas signal molecule delivery system can synergistically promote oxidation / nitration stress and sensitize the "ferroptosis" effect. According to the method of this embodiment, a NO gas signal molecule nano-delivery system can be obtained, which provides a new design idea and process flow for the research on gas-sensitized "ferroptosis" tumor treatment.

[0042] Specific embodiment 2: This embodiment differs from the specific embodiment 1 in that the preparation method of ZIF-8 is completed according to the following steps:

[0043] A mixed solution of 1 to 5 mmol of a zinc source and 10 to 20 mL of methanol was added to a 100 mL three-necked flask and stirred under N2 protection for 5 minutes. A mixed solution of 5 to 10 mmol of dimethylimidazole and 10 to 20 mL of methanol was then added, stirred for 1 to 3 hours, and centrifuged at 4000 rpm for 15 minutes. The solid material obtained after centrifugation was washed two to three times with methanol and finally dried to obtain ZIF-8. The zinc source was one or a mixture of zinc nitrate, zinc chloride, zinc acetate, and zinc acetylacetonate. The other steps were the same as those in the first embodiment.

[0044] Specific embodiment three: This embodiment differs from specific embodiment one or two in that the preparation method of ZIF-67 is completed according to the following steps:

[0045] 1 mL to 5 mL of a mixed aqueous solution of dimethylimidazole and hexadecyltrimethylammonium bromide is added to a round-bottom flask, stirred for 5 minutes, and then 1 mL to 5 mL of an aqueous solution of a cobalt source is added and stirred for 5 minutes to obtain a mixture; the mixture is allowed to stand at room temperature for 3 hours, and finally centrifuged at 4000 rpm for 15 minutes, and the solid matter obtained after centrifugation is washed 2 to 3 times with deionized water, and finally dried to obtain ZIF-67; the concentration of dimethylimidazole in the mixed aqueous solution of dimethylimidazole and hexadecyltrimethylammonium bromide is 500 mmol / L to 700 mmol / L, and the concentration of hexadecyltrimethylammonium bromide is 0.50 mmol / L to 2.00 mmol / L;

[0046] The concentration of the aqueous solution of the cobalt source is 50 mmol / L to 100 mmol / L, and the cobalt source is one or a mixture of cobalt nitrate, cobalt chloride and cobalt acetylacetonate. The other steps are the same as those of the first or second embodiment.

[0047] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that the preparation method of ZIF-90 is completed according to the following steps:

[0048] At 60°C, 0.5-1.0 mmol of dimethylimidazole was dissolved in 2-6 mL of dimethylformamide. A mixture of 0.1-0.3 mmol of a zinc source, 100-150 mg of polyvinylpyrrolidone, and 2-6 mL of dimethylformamide was then added. The mixture was stirred at room temperature for 5 minutes. 20 mL of dimethylformamide was then added and stirred for another 10 minutes. The mixture was then centrifuged at 4000 rpm for 15 minutes. The solid matter obtained after centrifugation was washed two to three times with methanol and dried to obtain ZIF-90. The zinc source was one or a mixture of zinc nitrate, zinc chloride, zinc acetate, and zinc acetylacetonate. The other steps were the same as those in Specific Embodiments 1 to 3.

[0049] Specific embodiment 5: This embodiment differs from specific embodiments 1 to 4 in that the volume ratio of the metal-organic framework material mass to water in step 1 is (0.01 g to 0.1 g):3 mL; and the volume ratio of the KMnO4 solution to water in step 1 is (0.01 mL to 0.1 mL):3 mL. The other steps are the same as specific embodiments 1 to 4.

[0050] Specific embodiment 6: The difference between this embodiment and specific embodiments 1 to 5 is that the concentration of the KMnO4 solution in step 1 is 1 mg mL -1In step 1, the metal-organic framework (MOF) is dissolved in water and stirred in an ice bath for 10 to 20 minutes. The KMnO₄ solution is then added and stirred in an ice bath for 30 to 120 minutes. Finally, the mixture is centrifuged at 4000 rpm for 10 to 20 minutes to obtain the ZIF@MnO₂ nanozyme. The remaining steps are the same as those in embodiments 1 to 5.

[0051] Specific Embodiment 7: This embodiment differs from Specific Embodiments 1 to 6 in that the NO donor in step 2 is L-arginine, S-nitrosothiol, S-nitroso-N-acetylpenicillamine, 4-nitro-3-trifluoromethylaniline, or an organic nitrate (glyceryl trinitrate and isosorbide mononitrate); and the mass ratio of the NO donor in step 2 to the volume of water is (0.05 mmol to 0.10 mmol):10 mL. The other steps are the same as Specific Embodiments 1 to 6.

[0052] Specific embodiment 8: This embodiment differs from specific embodiments 1 to 7 in that: in step 2, the mass ratio of the ZIF@MnO2 nanozyme to the volume of water is (10mg-20mg):10mL; the dark-proof stirring time in step 2 is 20h-24h; the washing is performed 2-3 times with deionized water; in step 2, the NO donor is first dissolved in water, and then the pH of the system is adjusted to 7.0 using 1mol / L hydrochloric acid. The other steps are the same as specific embodiments 1 to 7.

[0053] Specific embodiment 9: This embodiment differs from specific embodiments 1 to 8 in that the mass ratio of the ZIF@MnO2-NO nanodelivery system, polyethylene glycol, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide described in step 3 is (10 mg to 20 mg):(50 mg to 100 mg):15 mg. The other steps are the same as specific embodiments 1 to 8.

[0054] Specific embodiment 10: This embodiment differs from Specific embodiments 1 to 9 in that, in step 3, the ZIF@MnO2-NO nanodelivery system and polyethylene glycol are first mixed under ultrasound for 20 to 40 minutes, followed by the addition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, followed by stirring for 8 to 12 hours, and finally, centrifugation to collect the solid. The other steps are the same as Specific embodiments 1 to 9.

[0055] The following examples are used to verify the beneficial effects of the present invention:

[0056] Example 1: A method for synthesizing a nano-delivery system for NO gas signaling molecules to mediate reactive nitrogen / reactive oxygen species sensitization to the "ferroptosis" effect, specifically completed by the following steps:

[0057] 1. Synthesis of ZIF-67@MnO2 nanozyme:

[0058] First, 0.05 g of metal organic framework material was dissolved in 3 mL of water, then stirred for 10 min under ice bath conditions, and then 0.05 mL of 1 mg mL -1 The KMnO4 solution was stirred for 30 min in an ice bath and finally centrifuged at 4000 r / min for 10 min to obtain the ZIF-67@MnO2 nanozyme.

[0059] The metal organic framework material described in step 1 is ZIF-67; the preparation method of ZIF-67 is completed according to the following steps:

[0060] 2 mL of a mixed aqueous solution of dimethylimidazole and hexadecyltrimethylammonium bromide was added to a round-bottom flask and stirred for 5 minutes, followed by the addition of 2 mL of an aqueous solution of cobalt nitrate and stirring for 5 minutes to obtain a mixture; the mixture was allowed to stand at room temperature for 3 hours, and finally centrifuged at 4000 rpm for 15 minutes. The solid matter obtained after centrifugation was washed twice with deionized water and finally dried to obtain ZIF-67; the concentration of dimethylimidazole in the mixed aqueous solution of dimethylimidazole and hexadecyltrimethylammonium bromide was 500 mmol / L, and the concentration of hexadecyltrimethylammonium bromide was 0.60 mmol / L; the concentration of the aqueous solution of cobalt nitrate was 50 mmol / L;

[0061] 2. Synthesis of ZIF-67@MnO2-NO Nanodelivery System:

[0062] First, 0.05 mmol of NO donor L-arginine was dissolved in 10 mL of water. The pH of the system was then adjusted to 7.0 with 1 mol / L hydrochloric acid. 10 mg of ZIF-67@MnO2 nanozyme was then added. The mixture was stirred in the dark for 24 h, centrifuged, and the solid was collected and washed twice with deionized water to obtain the ZIF-67@MnO2-LA nanodelivery system.

[0063] 3. Synthesis of PEG / ZIF-67@MnO2-NO Nanodelivery System:

[0064] First, 10 mg of ZIF-67@MnO2-LA nanodelivery system was mixed with 50 mg of polyethylene glycol under ultrasound for 30 minutes, and then 15 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added. The mixture was stirred for 10 hours and the solid was collected by centrifugation to obtain the PEG / ZIF-67@MnO2-LA nanodelivery system, which is a nanodelivery system for the NO gas signaling molecule-mediated reactive nitrogen / reactive oxygen sensitization "ferroptosis" effect.

[0065] Example 2: This example differs from Example 1 in that the metal organic framework material in step 1 is ZIF-8; the preparation method of ZIF-8 is completed according to the following steps:

[0066] A mixed solution of 2 mmol of zinc source and 15 mL of methanol was added to a 100 mL three-necked flask, stirred for 5 min under N2 protection, and then a mixed solution of 7 mmol of dimethylimidazole and 13 mL of methanol was added, stirred for 2 h, and then centrifuged at 4000 r / min for 15 min. The solid material obtained after centrifugation was washed twice with methanol and finally dried to obtain ZIF-8; the zinc source was zinc nitrate.

[0067] Example 3: This example differs from Example 1 in that the metal organic framework material described in step 1 is ZIF-90, and the preparation method of ZIF-90 is completed according to the following steps:

[0068] At 60°C, 1.0 mmol of dimethylimidazole was dissolved in 5 mL of dimethylformamide, and a mixture of 0.2 mmol of a zinc source, 100 mg of polyvinyl pyrrolidone, and 5 mL of dimethylformamide was added. The mixture was stirred at room temperature for 5 minutes, and then 20 mL of dimethylformamide was added. The mixture was stirred for another 10 minutes, and finally centrifuged at 4000 r / min for 15 minutes. The solid material obtained after centrifugation was washed twice with methanol and finally dried to obtain ZIF-90; the zinc source was zinc nitrate.

[0069] Figure 1 TEM images of ZIF-8, ZIF-67, and ZIF-90, where a is ZIF-8 prepared in Example 2, b is ZIF-67 prepared in Example 1, and c is ZIF-90 prepared in Example 3;

[0070] from Figure 1 It can be seen that the ZIF-8, ZIF-67 and ZIF-90 nanoparticles are regular dodecahedron, cube and regular dodecahedron respectively.

[0071] Figure 2TEM, HAADF-STEM and element scanning images, in which a is the potassium permanganate oxidation mechanism diagram, b is the TEM image of ZIF-8@MnO2 solid before etching, c is the TEM image of ZIF-8@MnO2 hollow after etching, d is the HAADF-STEM and element scanning image of ZIF-8@MnO2, e is the TEM image of ZIF-67@MnO2 solid before etching, f is the TEM image of ZIF-67@MnO2 hollow after etching, g is the HAADF-STEM and element scanning image of ZIF-67@MnO2, h is the TEM image of ZIF-90@MnO2 solid before etching, i is the TEM image of ZIF-90@MnO2 hollow after etching, and j is the HAADF-STEM and element scanning image of ZIF-90@MnO2;

[0072] The MnO2 shell was in situ grown on the ZIFs skeleton via redox reaction, and potassium permanganate oxidized the methyl groups on 2-methylimidazole and imidazole-2-carboxaldehyde to carboxyl groups ( Figure 2 a), MnO2 is attached to the surface of the ZIFs skeleton. At the same time, the oxidized carboxylic acid can release protons (H+), and by controlling the reaction time, the solid ZIF structure is etched into a hollow structure. Figure 2 b and c are TEM images of solid and hollow ZIF-8 before and after etching, respectively. The hollow structure can be clearly seen. Figure 2 d is the HAADF-STEM and elemental scanning of ZIF-8@MnO2. ZIF-8@MnO2 is composed of Mn, Zn, C, N and O. Similarly, the TEM and elemental scanning of ZIF-67@MnO2 and ZIF-67@MnO2 also confirmed the hollow structure and the composition of Mn, Co, C, N and O. Figure 2 Eg); Similarly, the TEM and element scanning images of ZIF-90@MnO2 and ZIF-90@MnO2 also confirmed the hollow structure and the composition of elements such as Mn, Co, C, N and O ( Figure 2 hj);

[0073] Figure 3 XRD patterns, in which (a) is the XRD pattern of ZIF-8, ZIF-8@MnO2 solid before etching (ZIF-8-S@MnO2) and ZIF-8@MnO2 hollow after etching (ZIF-8-H@MnO2), (b) is the XRD pattern of ZIF-67, ZIF-67@MnO2 solid before etching (ZIF-67@MnO2) and ZIF-67@MnO2 hollow after etching (ZIF-67@MnO2), and (c) is the XRD pattern of ZIF-90, ZIF-90@MnO2 solid before etching and ZIF-90@MnO2 hollow after etching;

[0074] from Figure 3It can be seen that the angles of all diffraction peaks are consistent with the standard card of ZIFs structure. This shows that the method of using potassium permanganate to oxidize methyl or aldehyde groups to carboxyl groups and then reducing them to MnO2 and etching them into hollow structures by controlling the reaction time is feasible. Comparing three nanozymes with similar structures, according to the literature, the ability of Co element to produce ·OH through Fenton is better than that of Mn. 2+ , the ROS generated is high, which is conducive to the realization of oxidative stress-mediated "ferroptosis". Therefore, ZIF-67@MnO2 was selected as a carrier for material transportation, loaded with L-arginine and then coated with PEG to improve biocompatibility. The final product PEG / ZIF-67@MnO2-LA was used for subsequent performance studies;

[0075] Figure 4 The full XPS spectrum of PEG / ZIF-67@MnO2-LA and the high-resolution XPS spectra of Mn 2p and Co 2p;

[0076] The full XPS spectrum shows that PEG / ZIF-67@MnO2-LA is composed of Mn, Co, N, O and C. The high-resolution XPS spectrum of Mn 2p shows two obvious peaks with a spin energy separation of 11.7 eV, namely Mn 2p3 / 2 (642.7 eV) and Mn 2p1 / 2 (654.4 eV). According to the fitting curve, the peaks at 653.27 eV and 641.71 eV belong to Mn 3+ , the peaks at 654.65eV and 643.03eV belong to Mn 4+ ;

[0077] Figure 5 N2 adsorption-desorption curves of ZIF-67 and ZIF-67@MnO2, where 1 is ZIF-67 and 2 is ZIF-67@MnO2;

[0078] The specific surface areas of ZIF-67 and ZIF-67@MnO2 analyzed by Brunauer-Emmett-Teller (BET) method were 1178.31 m 2 g -1 and 649.64m 2 g -1 The large specific surface area of ​​ZIF-67@MnO2 nanozyme is conducive to the loading of LA by electrostatic interaction.

[0079] Figure 6 Zeta potential diagrams of ZIF-67, ZIF-67@MnO2, ZIF-67@MnO2-LA and PEG / ZIF-67@MnO2-LA;

[0080] The change in zeta potential confirmed the successful loading of L-arginine and the successful synthesis of PEG / ZIF-67@MnO2-LA nanozyme.

[0081] Figure 7 pH and GSH response decomposition curve of PEG / ZIF-67@MnO2-LA;

[0082] PEG / ZIF-67@MnO2-LA was dispersed in phosphate buffered saline (PBS) containing GSH (2mM and 5mM) and without GSH at pH 6.5 and 7.4 for 90 minutes. After incubation for 90 minutes in the absence of GSH at pH 7.4, the PEG / ZIF-67@MnO2-LA nanozyme maintained a relatively intact structure, while significant decomposition occurred in the presence of GSH at pH 6.5. The results showed that the PEG / ZIF-67@MnO2-LA nanozyme degraded into small clusters after 90 minutes under the conditions of 5mM GSH at pH 6.5. The PEG / ZIF-67@MnO2-LA nanozyme has good responsiveness to pH and GSH and has good prospects for in vivo biological applications;

[0083] Figure 8 Figure 2 is the degradation transmission photo of PEG / ZIF-67@MnO2-LA under different GSH conditions and the percentage of GSH consumed. Figure a is the degradation transmission photo, b is the mechanism diagram of DTNB detection of GSH, and c is the percentage of GSH consumed by different concentrations of PEG / ZIF-67@MnO2-LA.

[0084] Under different pH conditions, GSH release triggered different degradation of PEG / ZIF-67@MnO2-LA. At pH = 6.5 and GSH = 5mM, PEG / ZIF-67@MnO2-LA was completely degraded in 90 minutes. GSH consumption was assessed using a 5,5'-dithiothio(2-nitrobenzoic acid) (DTNB) probe, which specifically recognizes sulfhydryl groups at an absorption peak of 412nm. PEG / ZIF-67@MnO2-LA nanozymes consumed more GSH when the concentration and / or reaction time were increased.

[0085] Figure 9 To explore the CAT enzyme activity of PEG / ZIF-67@MnO2-LA, Figure a is the dissolved oxygen curve of different solutions, and b is the Michaelis-Menten fitting curve under different conditions;

[0086] Figure 9In the dissolved oxygen curves of different solutions in a, H2O2 and H2O alone can produce a small amount of O2. ZIF-67 and MnO2 also have some CAT-like activity and produce some O2. In comparison, PEG / ZIF-67@MnO2-LA nanozyme produces the most O2, which means it has the strongest ability to decompose H2O2 and the best CAT-like activity. Figure 9 b is the Michaelis-Menten fitting curve under different conditions.

[0087] Figure 10 To explore the generation of ·OH in PEG / ZIF-67@MnO2-LA;

[0088] Regarding the ability of PEG / ZIF-67@MnO2-LA nanozyme to catalyze H2O2 to produce ·OH, methylene blue (MB) was used as a probe, and the changes in the UV absorption-visible spectrum of MB before and after the reaction were used to qualitatively determine the ability to produce ·OH. Compared with the other four control groups, MB+PEG / ZIF-67@MnO2-LA+H2O2+HCO3 - The UV absorption curve of the group decreased most significantly, indicating that most of the MB had been degraded by the generated ·OH, and also indicating that the PEG / ZIF-67@MnO2-LA nanozyme - Under the conditions of its existence, H2O2 can be decomposed to produce ·OH, and more ·OH is produced as time increases.

[0089] Figure 11 To investigate the OXD enzyme activity of PEG / ZIF-67@MnO2-LA;

[0090] The OXD-like enzyme activity of PEG / ZIF-67@MnO2-LA nanozyme at pH 6.5 and 7.4 was studied using TMB as substrate. PEG / ZIF-67@MnO2-LA can react with O2 in the solution to produce ·O2 - The blue TMB solution faded and the UV absorption value at 650 nm decreased. At pH 6.5, PEG / ZIF-67@MnO2-LA produced ·O2 - The reaction is faster and easier to proceed than when the pH is 7.4. Finally, the ESR method was used to test the O2 - signals, such as Figure 11 As shown in a, at pH 7.4, O2 - The signal is very weak; at pH 6.5, the signal is enhanced, and when H2O2 is added, the signal is the strongest. This is because the CAT-like activity increases the content of O2 in the solution, so O2 - The output also increases, and the signal is the strongest. Figure 11b is the Michaelis-Menten fitting curve of PEG / ZIF-67@MnO2-LA under different conditions.

[0091] Figure 12 To investigate the NO release performance of PEG / ZIF-67@MnO2-LA;

[0092] The release of NO was studied using a Griess kit in a reducing environment. Under normal conditions, PEG / ZIF-67@MnO2-LA did not release any significant NO. However, in the presence of H2O2, NO was significantly released. The released NO could also react with the generated O2 - The reaction generates the more toxic ONOO - Next, L-tyrosine was used as a fluorescent probe to determine the production of ONOO- by observing the fluorescence emission intensity at 406 nm. The ZHMAP group had a fluorescence intensity, indicating the presence of ONOO - Generation.

[0093] Figure 13 To explore the therapeutic properties of PEG / ZIF-67@MnO2-LA at the cellular level;

[0094] exist Figure 13 In the experiment, the cells in the control group were almost all green fluorescent, indicating that the cells grew normally under normal conditions; the cells in the ZIF-67 and IF-67@MnO2 groups had both red and green fluorescent light, indicating that ZIF-67 and IF-67@MnO2 were toxic to cells and could kill some cells; in the PEG / ZIF-67@MnO2-LA group, almost no green normally growing cells were observed, and the cells were almost all red, indicating that the cell toxicity was the greatest.

Claims

1. A method for synthesizing a nano-delivery system for NO gas signaling molecules to mediate reactive nitrogen and reactive oxygen species sensitizing ferroptosis, characterized in that: The synthetic method is specifically completed according to the following steps:

1. Synthesis of ZIF@MnO2 nanozymes: First, the metal organic framework material was dissolved in water, then stirred in an ice bath, KMnO4 solution was added, and stirring was continued in an ice bath, and finally centrifuged to obtain ZIF@MnO2 nanozyme; The metal organic framework material described in step 1 is ZIF-8, ZIF-67 or ZIF-90; 2. Synthesis of ZIF@MnO2-NO Nanodelivery System: First, the NO donor was dissolved in water, and then the pH value of the system was adjusted to 7.

0. Then, the ZIF@MnO2 nanozyme was added, stirred in the dark, centrifuged, and the solid was collected and washed to obtain the ZIF@MnO2-NO nanodelivery system. The NO donor in step 2 is L-arginine, S-nitrosothiol, S-nitroso-N-acetylpenicillamine, 4-nitro-3-trifluoromethylaniline or an organic nitrate; the organic nitrate is glyceryl trinitrate or isosorbide mononitrate; 3. Synthesis of PEG / ZIF@MnO2-NO Nanodelivery System: First, the ZIF@MnO2-NO nanodelivery system was mixed with polyethylene glycol under ultrasound, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added. The reaction was stirred and finally centrifuged and the solid was collected to obtain the PEG / ZIF@MnO2-NO nanodelivery system, which is a nanodelivery system for the NO gas signal molecule-mediated reactive nitrogen and reactive oxygen species-sensitized ferroptosis effect.

2. The method for synthesizing a nano-delivery system for the NO gas signaling molecule-mediated reactive nitrogen and reactive oxygen species-sensitized ferroptosis effect according to claim 1, characterized in that: The preparation method of ZIF-8 is completed according to the following steps: A mixed solution of 1 mmol to 5 mmol of a zinc source and 10 mL to 20 mL of methanol is added to a 100 mL three-necked flask, stirred for 5 minutes under N2 protection, and then a mixed solution of 5 mmol to 10 mmol of dimethylimidazole and 10 mL to 20 mL of methanol is added, stirred for 1 hour to 3 hours, and then centrifuged at 4000 r / min for 15 minutes. The solid matter obtained after centrifugation is washed 2 to 3 times with methanol, and finally dried to obtain ZIF-8; the zinc source is one or a mixture of zinc nitrate, zinc chloride, zinc acetate and zinc acetylacetonate.

3. The method for synthesizing a nano-delivery system for the NO gas signaling molecule-mediated reactive nitrogen and reactive oxygen species-sensitized ferroptosis effect according to claim 1, characterized in that: The preparation method of ZIF-67 is completed according to the following steps: 1 mL to 5 mL of a mixed aqueous solution of dimethylimidazole and hexadecyltrimethylammonium bromide was added to a round-bottom flask, stirred for 5 minutes, and then 1 mL to 5 mL of an aqueous solution of a cobalt source was added and stirred for 5 minutes to obtain a mixture; the mixture was allowed to stand at room temperature for 3 hours, and finally centrifuged at 4000 r / min for 15 minutes, and the solid matter obtained after centrifugation was washed 2 to 3 times with deionized water, and finally dried to obtain ZIF-67; the concentration of dimethylimidazole in the mixed aqueous solution of dimethylimidazole and hexadecyltrimethylammonium bromide was 500 mmol / L to 700 mmol / L, and the concentration of hexadecyltrimethylammonium bromide was 0.50 mmol / L to 2.00 mmol / L; The concentration of the aqueous solution of the cobalt source is 50 mmol / L to 100 mmol / L, and the cobalt source is one or a mixture of cobalt nitrate, cobalt chloride and cobalt acetylacetonate.

4. The method for synthesizing a nano-delivery system for the NO gas signaling molecule-mediated reactive nitrogen and reactive oxygen species-sensitized ferroptosis effect according to claim 1, characterized in that: The preparation method of ZIF-90 is completed according to the following steps: At 60°C, 0.5mmol~1.0mmol dimethylimidazole is dissolved in 2mL~6mL dimethylformamide, and then a mixture of 0.1mmol~0.3mmol zinc source, 100mg~150mg polyvinylpyrrolidone and 2mL~6mL dimethylformamide is added. The mixture is stirred at room temperature for 5 minutes, and then 20mL dimethylformamide is added. The mixture is stirred for another 10 minutes, and finally centrifuged at 4000r / min for 15 minutes. The solid matter obtained after centrifugation is washed 2~3 times with methanol and finally dried to obtain ZIF-90. The zinc source is one or a mixture of zinc nitrate, zinc chloride, zinc acetate and zinc acetylacetonate.

5. The method for synthesizing a nano-delivery system for the NO gas signaling molecule-mediated reactive nitrogen and reactive oxygen species-sensitized ferroptosis effect according to claim 1, characterized in that: The mass ratio of the metal organic framework material described in step 1 to the volume ratio of water is (0.01g~0.10g):3mL; the volume ratio of the KMnO4 solution described in step 1 to the volume ratio of water is (0.01mL~0.1mL):3mL.

6. The method for synthesizing a nano-delivery system for the NO gas signaling molecule-mediated reactive nitrogen and reactive oxygen species-sensitized ferroptosis effect according to claim 1, characterized in that: The concentration of the KMnO4 solution described in step one is 1 mg / mL; in step one, the metal organic framework material is first dissolved in water, then stirred in an ice bath for 10 min to 20 min, the KMnO4 solution is added, and the stirring is continued in an ice bath for 30 min to 120 min, and finally centrifuged at 4000 r / min for 10 min to 20 min to obtain the ZIF@MnO2 nanozyme.

7. The method for synthesizing a nano-delivery system for the NO gas signaling molecule-mediated reactive nitrogen and reactive oxygen species-sensitized ferroptosis effect according to claim 1, characterized in that: The mass ratio of the NO donor described in step 2 to the volume of water is (0.05 mmol ~ 0.10 mmol): 10 mL.

8. The method for synthesizing a nano-delivery system for the NO gas signaling molecule-mediated reactive nitrogen and reactive oxygen species-sensitized ferroptosis effect according to claim 1, characterized in that: The mass ratio of the ZIF@MnO2 nanozyme described in step 2 to the volume ratio of water is (10 mg~20 mg):10 mL; the time of stirring in the dark described in step 2 is 20 h~24 h; the washing is performed using deionized water for 2~3 times; in step 2, the NO donor is first dissolved in water, and then the pH value of the system is adjusted to 7.0 using 1 mol / L hydrochloric acid.

9. The method for synthesizing a nano-delivery system for the NO gas signaling molecule-mediated reactive nitrogen and reactive oxygen species-sensitized ferroptosis effect according to claim 1, characterized in that: The mass ratio of the ZIF@MnO2-NO nanodelivery system, polyethylene glycol, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide described in step 3 is (10 mg~20 mg):(50 mg~100 mg):15 mg.

10. The method for synthesizing a nano-delivery system for the NO gas signaling molecule-mediated reactive nitrogen and reactive oxygen species-sensitized ferroptosis effect according to claim 1, characterized in that: In step 3, the ZIF@MnO2-NO nanodelivery system was first mixed with polyethylene glycol under ultrasound for 20 min to 40 min, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added. The mixture was stirred for 8 h to 12 h, and finally centrifuged to collect the solid.

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