Multi-response type nano gas therapeutic agent as well as preparation method and application thereof

Abstract

The invention relates to a multi-response type nano gas therapeutic agent as well as a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations. The gas therapeutic agent comprises MnCO nanoparticles coated with a PLGA-SNAP polymer, the MnCO nanoparticles can respond to GSH and H2O2 which are overexpressed in tumor cells to generate double gases (NO and CO), and NO and CO can effectively induce mitochondrial damage through a synergistic effect and generate peroxynitrite (ONOO-) through a cascade reaction to further induce cell nucleus damage. Meanwhile, mtDNA and gDNA released by damaged mitochondria and cell nucleuses can activate cGAS / STING pathways in DCs cells, induce the cGAS / STING pathways to be mature and stimulate secretion of IFN-beta, and start of effector T cells in the tumor microenvironment is enhanced. In addition, the gas therapeutic agent does not cause side effects on normal cells or tissues.

Description

Technical Field

[0001] The present application relates to the technical field of pharmaceutical preparations, and in particular to a multi-responsive nano gas therapeutic agent and a preparation method and application thereof. Background Art

[0002] Cancer, as a disease with a high mortality rate, seriously threatens human health. At present, clinical treatments for cancer mainly include surgical resection, radiotherapy and chemotherapy, but they often have certain toxic side effects and are prone to recurrence and metastasis. It is urgent to develop safe and effective treatment strategies. In recent years, immunotherapy has attracted widespread attention because of its ability to effectively activate the immune system to identify, attack and eliminate tumor cells. Among them, immune checkpoint inhibitors (such as programmed death receptor 1 (PD-1) / programmed death ligand 1 (PD-L1) inhibitors) have shown good immunotherapy effects in anti-tumor treatment. However, due to the complex immunosuppressive tumor microenvironment and individual differences in cancer patients, these immune checkpoint inhibitors are not always effective. Therefore, how to enhance the responsiveness of cancer patients to immunotherapy is an urgent problem to be solved. As a newly discovered immunotherapy pathway, the cyclic GMP-AMP synthase / stimulator of interferon genes (cGAS / STING) pathway has become a promising method to promote cancer immunotherapy. It is cGAS in dendritic cells (DCs) that can sense cytoplasmic dsDNA (double-stranded DNA) to activate the STING pathway and induce the secretion of type I interferon (IFN-β), promoting the infiltration of CD4+ / CD8+T cells in tumor tissues. Therefore, how to improve the activation effect of the cGAS / STING signaling pathway in tumor tissues is particularly critical.

[0003] As an emerging treatment method, gas therapy can use gas molecules to induce cancer cell death and achieve good therapeutic effects, which has attracted widespread attention. It is reported that high concentrations of CO and NO can directly kill cancer cells through multiple pathways such as generating oxidative stress, damaging mitochondria, inhibiting DNA synthesis / repair, inhibiting energy metabolism of tumor cells, enhancing inflammatory response and promoting immune response. In addition, damaged mitochondria and cell nuclei can also release mitochondrial DNA and nuclear DNA, which has the potential to activate the cGAS / STING signaling pathway. However, the low solubility, high diffusivity and strong stimulation of respiratory tissues of these gas molecules have greatly limited their application in actual treatment. Summary of the invention

[0004] Based on the existing problems of tumor immunotherapy and gas therapy, the inventors found that the mitochondrial damage was caused by the synergistic effect of CO and NO gas molecules, and the peroxynitrite (ONOO -) further damages the nucleus of tumor cells. The damaged mitochondria and nucleus release double-stranded deoxyribonucleic acid (dsDNA), which can activate the cyclic guanosine monophosphate-adenylate synthetase (cGAS)-stimulator of interferon genes (STING) pathway in dendritic cells (DCs) and induce the secretion of interferon-β (IFN-β), thereby effectively promoting the maturation of DCs and enhancing the immune response.

[0005] The present application provides a multi-responsive nano gas therapeutic agent and a preparation method and application thereof to coordinate gas therapy and immunotherapy.

[0006] In a first aspect, the present application provides a multi-responsive nano gas therapeutic agent, wherein the gas therapeutic agent comprises nanoparticles of MnCO coated with a PLGA-SNAP polymer.

[0007] As an optional embodiment, in the nanoparticles, the mass ratio of PLGA-SNAP polymer to MnCO is 10:(1.5-3).

[0008] As an optional implementation manner, the particle size of the nanoparticles is 80 to 250 nm.

[0009] In a second aspect, the present application provides a method for preparing a multi-responsive nano gas therapeutic agent, the method comprising:

[0010] The amino-terminated poly(lactic acid-co-glycolic acid) and S-nitroso-N-acetyl-DL-penicillamine are subjected to an amidation reaction to graft S-nitroso-N-acetyl-DL-penicillamine onto the poly(lactic acid-co-glycolic acid) to obtain a PLGA-SNAP polymer;

[0011] The PLGA-SNAP polymer is coated on MnCO by an emulsification-solvent evaporation method to obtain a gas therapeutic agent.

[0012] As an optional embodiment, the amidation reaction of the amino-terminated poly(lactic acid-co-glycolic acid) and S-nitroso-N-acetyl-DL-penicillamine to graft S-nitroso-N-acetyl-DL-penicillamine onto the poly(lactic acid-co-glycolic acid) to obtain the PLGA-SNAP polymer comprises:

[0013] The amino-terminated polylactic acid-glycolic acid copolymer, a condensation agent, an activator and S-nitroso-N-acetyl-DL-penicillamine are mixed and dissolved in a first solvent, and then an amidation reaction is performed to graft S-nitroso-N-acetyl-DL-penicillamine onto the polylactic acid-glycolic acid copolymer to obtain a PLGA-SNAP polymer.

[0014] As an optional embodiment, the condensation agent includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0015] As an optional embodiment, the activator includes N-hydroxysuccinimide.

[0016] As an optional embodiment, the first solvent includes dimethyl sulfoxide.

[0017] As an optional embodiment, the method of coating the PLGA-SNAP polymer on MnCO by an emulsification-solvent evaporation method to obtain a gas therapeutic agent comprises:

[0018] Dissolving the PLGA-SNAP polymer in a second solvent, and then mixing with MnCO to obtain an organic phase;

[0019] The organic phase and the bovine serum albumin aqueous solution are mixed and ultrasonically emulsified to obtain an emulsion;

[0020] The emulsion and deionized water are mixed and stirred, and then the organic solvent is evaporated to obtain a gas therapeutic agent.

[0021] As an optional embodiment, the second solvent includes dichloromethane.

[0022] In a third aspect, the present application provides an application of a multi-responsive nanogas therapeutic agent, which includes using the multi-responsive nanogas therapeutic agent provided in the first aspect or the multi-responsive nanogas therapeutic agent prepared by the method provided in the second aspect for the preparation of a drug.

[0023] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0024] The gas therapeutic agent provided in the embodiment of the present application can produce dual gases (NO and CO) in response to the overexpression of GSH and H2O2 in tumor cells to induce mitochondrial damage, and produce peroxynitrite (ONOO) through a cascade reaction. - ) further induces nuclear damage. At the same time, the mtDNA and gDNA released by damaged mitochondria and nuclei can activate the cGAS / STING pathway in DCs cells and induce their maturation and stimulate the secretion of IFN-β, enhancing the activation of effector T cells in the tumor microenvironment. In addition, the gas therapy agent will not have side effects on normal cells or tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0026] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0027] Figure 1 A schematic diagram of a process for a method provided in an embodiment of the present application;

[0028] Figure 2 The PLGA-SNAP polymer provided in the embodiment of the present application 1 H NMR spectrum;

[0029] Figure 3 a is a TEM image of the gas therapeutic agent provided in Example 2 of the present application, b is a SEM image of the gas therapeutic agent provided in Example 2 of the present application, c is a DLS result image of the gas therapeutic agent provided in Example 2 of the present application, and d is a Zeta potential image of the gas therapeutic agent provided in Example 2 of the present application;

[0030] Figure 4 Fluorescence images of CO and NO after incubation of HUVEC cells with the gaseous therapeutic agent provided in Example 2 of the present application for different periods of time;

[0031] Figure 5 Fluorescence images of CO and NO after incubation of the gas therapeutic agent provided in Example 2 of the present application with 4T1 cells for different periods of time;

[0032] Figure 6 A graph showing the results of a study on cellular phagocytosis and lysosomal escape of a gaseous therapeutic agent provided in an embodiment of the present application;

[0033] Figure 7 This is a result diagram of a study on the induction of DCs maturation and IFN-β secretion by gaseous therapeutic agents provided in Example 2 of the present application;

[0034] Figure 8 This is a graph showing the results of investigating the in vivo anti-tumor effect of the gas therapeutic agent provided in Example 2 of the present application. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0036] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0037] Currently, gas therapy and immunotherapy face the following problems: the low solubility, high diffusivity, and strong stimulation of respiratory tissues of gas molecules have greatly limited the application of gas therapy in actual treatment; gas prodrugs are mostly small molecule compounds with short circulation time in the body and lack the ability to actively aggregate and controllably release in tumors, resulting in unsatisfactory treatment effects; due to the complex immunosuppressive tumor microenvironment (ITM) and individual differences among cancer patients, immune checkpoint inhibitors are not always effective.

[0038] Based on the current problems with tumor immunotherapy and gas therapy, the inventors of the present application realized that the synergistic effect of CO and NO gas molecules can cause mitochondrial damage, and the cascade reaction of the two can be used to destroy the nucleus of tumor cells. The damaged mitochondria and nuclei release double-stranded deoxyribonucleic acid (dsDNA), which can activate the cyclic guanosine monophosphate-adenylate synthetase (cGAS)-stimulator of interferon genes (STING) pathway in dendritic cells (DCs) and induce the secretion of interferon-β (IFN-β), thereby effectively promoting the maturation of DCs and enhancing the immune response.

[0039] Based on this, the present application aims to develop an emerging therapeutic method by combining prodrugs with nanocarriers, which uses endogenous stimulation to achieve tumor-targeted delivery and controllable release of gas molecules, induce apoptosis of cancer cells by destroying their redox homeostasis, promote DCs maturation, activate immune response and improve the effect of immunotherapy.

[0040] The embodiment of the present application provides a multi-responsive nano gas therapeutic agent, which includes nanoparticles of MnCO coated with PLGA-SNAP polymer.

[0041] The gas therapeutic agent can produce dual gases (NO and CO) in response to the overexpression of GSH and H2O2 in tumor cells, inducing mitochondrial damage and generating peroxynitrite (ONOO) through a cascade reaction. - ) further induces nuclear damage. Damaged mitochondria and nuclei release double-stranded deoxyribonucleic acid (dsDNA), which can activate the cGAS / STING pathway in DCs cells and induce their maturation and stimulate the secretion of IFN-β, enhancing the activation of effector T cells in the tumor microenvironment. In addition, the gas therapy agent does not have side effects on normal cells or tissues.

[0042] In some embodiments, in the nanoparticles, the mass ratio of PLGA-SNAP polymer to MnCO is 10:

[0043] (1.5-3). Preferably, the mass ratio of PLGA-SNAP polymer to MnCO is 10:2.

[0044] In some embodiments, the nanoparticles have a particle size of 80 to 250 nm.

[0045] Figure 1 A flow chart of the method provided in the embodiment of the present application is as follows: Figure 1 As shown, based on a general inventive concept, the present application embodiment also provides a method for preparing a multi-responsive nano gas therapeutic agent, the method comprising:

[0046] S1. An amino-terminated poly(lactic acid-co-glycolic acid) (PLGA-NH2) and S-nitroso-N-acetyl-DL-penicillamine (SNAP) are subjected to an amidation reaction to graft S-nitroso-N-acetyl-DL-penicillamine onto the poly(lactic acid-co-glycolic acid) to obtain a PLGA-SNAP polymer;

[0047] In some embodiments, the amidation reaction of amino-terminated poly(lactic acid-co-glycolic acid) (PLGA-NH2) and S-nitroso-N-acetyl-DL-penicillamine (SNAP) to graft S-nitroso-N-acetyl-DL-penicillamine onto poly(lactic acid-co-glycolic acid) to obtain a PLGA-SNAP polymer comprises:

[0048] An amino-terminated poly(lactic acid-glycolic acid) copolymer (PLGA-NH2), a condensation agent, an activator and S-nitroso-N-acetyl-DL-penicillamine (SNAP) are mixed and dissolved in a first solvent, and then an amidation reaction is performed to graft S-nitroso-N-acetyl-DL-penicillamine (SNAP) onto the poly(lactic acid-glycolic acid) copolymer (PLGA) to obtain a PLGA-SNAP polymer.

[0049] The condensing agent includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, the activating agent includes N-hydroxysuccinimide, and the first solvent includes dimethyl sulfoxide.

[0050] Specifically, in this embodiment, polylactic acid-co-glycolic acid linked amino group (PLGA-NH2) is dissolved in the first solvent and mixed and stirred for 4h, and a condensing agent and an activator are added respectively under stirring, and finally S-nitroso-N-acetyl-DL-penicillamine (SNAP) is added, and the stirring reaction is continued in the dark (room temperature). After the reaction is completed, the reacted solution is thoroughly dialyzed with deionized water in the dark for 3 days (MWCO 1000). Finally, the product solution is freeze-dried and stored in a dark place.

[0051] S2. The PLGA-SNAP polymer is coated on MnCO by an emulsification-solvent evaporation method to obtain a gas therapeutic agent.

[0052] In some embodiments, the method of coating the PLGA-SNAP polymer on MnCO by an emulsification-solvent evaporation method to obtain a gas therapeutic agent comprises:

[0053] S2.1. Dissolve the PLGA-SNAP polymer in a second solvent, and then mix with MnCO to obtain an organic phase; wherein the second solvent includes dichloromethane.

[0054] S2.2. The organic phase and the aqueous solution of bovine serum albumin are mixed and ultrasonically emulsified to obtain an emulsion;

[0055] S2.3. The emulsion and deionized water are mixed and stirred, and then the organic solvent is evaporated to obtain a gas therapeutic agent.

[0056] Specifically, in this embodiment, PLGA-SNAP polymer is dissolved in a second solvent to form a solution. Carbonyl manganese (MnCO) is dissolved in the above solution to form an organic phase. The organic phase is then quickly poured into a bovine serum albumin (BSA) aqueous solution and ultrasonically emulsified to obtain an emulsion. Finally, the emulsion is poured into deionized water and stirred at room temperature to completely evaporate the organic solvent. Finally, centrifugation is performed to obtain a gas therapeutic agent (MnCO-SNAP@P nanoparticles).

[0057] Based on the same inventive concept, an embodiment of the present application also provides an application of a multi-responsive nano-gas therapeutic agent, which includes using the multi-responsive nano-gas therapeutic agent provided above or the multi-responsive nano-gas therapeutic agent prepared by the method provided above for the preparation of drugs.

[0058] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0059] Example 1

[0060] A multi-responsive nano gas therapeutic agent, the preparation method of which is as follows:

[0061] 1. Dissolve 10 mg of polylactic acid-co-glycolic acid linked amino group (PLGA-NH2) in 15 mL of dimethyl sulfoxide (DMSO) solvent and stir for 4 h. Add 30 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and 36 mg of N-hydroxysuccinimide (NHS) respectively under stirring, and finally add 2 mg of S-nitroso-N-acetyl-DL-penicillamine (SNAP). Continue stirring and reacting in the dark for 24 h (room temperature). After the reaction is completed, the solution after the reaction is thoroughly dialyzed with deionized water in the dark for 3 days (MWCO 1000). Finally, the product solution is freeze-dried and stored in a dark place.

[0062] 2. Dissolve 10 mg of the prepared PLGA-SNAP polymer in 1 mL of dichloromethane (DCM) to form a solution. Dissolve 1 mg of carbonyl manganese (MnCO) in the above solution to form an organic phase. Then quickly pour the organic phase into 4 mL of bovine serum albumin (BSA) aqueous solution (3%, w / v), ultrasonically emulsify for 60 seconds to obtain an emulsion. Finally, pour the emulsion into 20 mL of deionized water and stir at room temperature for 4 hours to completely evaporate the organic solvent. Finally, centrifuge at 12,000 rpm for 10 minutes to obtain a gas therapeutic agent.

[0063] Example 2

[0064] A multi-responsive nano gas therapeutic agent, the preparation method of which is as follows:

[0065] 1. Dissolve 10 mg of polylactic acid-co-glycolic acid linked amino group (PLGA-NH2) in 15 mL of dimethyl sulfoxide (DMSO) solvent and stir for 4 h. Add 30 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and 36 mg of N-hydroxysuccinimide (NHS) respectively under stirring, and finally add 2 mg of S-nitroso-N-acetyl-DL-penicillamine (SNAP). Continue stirring and reacting in the dark for 24 h (room temperature). After the reaction is completed, the solution after the reaction is thoroughly dialyzed with deionized water in the dark for 3 days (MWCO 1000). Finally, the product solution is freeze-dried and stored in a dark place.

[0066] 2. Dissolve 10 mg of the prepared PLGA-SNAP polymer in 1 mL of dichloromethane (DCM) to form a solution. Dissolve 2 mg of carbonyl manganese (MnCO) in the above solution to form an organic phase. Then quickly pour the organic phase into 4 mL of bovine serum albumin (BSA) aqueous solution (3%, w / v), ultrasonically emulsify for 60 seconds to obtain an emulsion. Finally, pour the emulsion into 20 mL of deionized water and stir at room temperature for 4 hours to completely evaporate the organic solvent. Finally, collect by centrifugation at 12,000 rpm for 10 minutes to obtain a gas therapeutic agent.

[0067] Example 3

[0068] A multi-responsive nano gas therapeutic agent, the preparation method of which is as follows:

[0069] 1. Dissolve 10 mg of polylactic acid-co-glycolic acid linked amino group (PLGA-NH2) in 15 mL of dimethyl sulfoxide (DMSO) solvent and stir for 4 h. Add 30 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and 36 mg of N-hydroxysuccinimide (NHS) respectively under stirring, and finally add 2 mg of S-nitroso-N-acetyl-DL-penicillamine (SNAP). Continue stirring and reacting in the dark for 24 h (room temperature). After the reaction is completed, the solution after the reaction is thoroughly dialyzed with deionized water in the dark for 3 days (MWCO 1000). Finally, the product solution is freeze-dried and stored in a dark place.

[0070] 2. Dissolve 10 mg of the prepared PLGA-SNAP polymer in 1 mL of dichloromethane (DCM) to form a solution. Dissolve 4 mg of carbonyl manganese (MnCO) in the above solution to form an organic phase. Then quickly pour the organic phase into 4 mL of bovine serum albumin (BSA) aqueous solution (3%, w / v), ultrasonically emulsify for 60 seconds to obtain an emulsion. Finally, pour the emulsion into 20 mL of deionized water and stir at room temperature for 4 hours to completely evaporate the organic solvent. Finally, centrifuge at 12,000 rpm for 10 minutes to obtain a gas therapeutic agent.

[0071] Experimental example

[0072] The PLGA-SNAP polymers prepared in each embodiment were 1 H NMR characterization, the results are as follows Figure 2 As shown, Figure 2 The PLGA-SNAP polymer provided in the embodiment of the present application 1 H NMR spectrum, from which we can get, 1 The appearance of amide hydrogen peak in the H NMR spectrum confirmed the successful synthesis of PLGA-SNAP polymer.

[0073] The killing effect of the gas therapeutic agents provided in Examples 1 to 4 on 4T1 cells was determined using the Alamar Blue (AB) method; the results are shown in the following table:

[0074]

[0075] From the above table, it can be seen that when the mass ratio of PLGA-SNAP polymer to MnCO is 10:2-10:4, CI 50 The value of is controlled below 1, indicating that SNAP and MnCO have a good synergistic effect, especially when the mass ratio of PLGA-SNAP polymer to MnCO is 10:2, CI 50 The value of is 0.81.

[0076] The gas therapeutic agent provided in Example 2 was subjected to morphological characterization: transmission electrolysis test, scanning electrolysis test, DLS test and Zeta potential test were performed respectively. The results are as follows: Figure 3 As shown, Figure 3 In a, the TEM image of the gas therapeutic agent provided in Example 2 of the present application, b, the SEM image of the gas therapeutic agent provided in Example 2 of the present application, c, the DLS result image of the gas therapeutic agent provided in Example 2 of the present application, and d, the Zeta potential image of the gas therapeutic agent provided in Example 2 of the present application. From the TEM and SEM images, it can be seen that the MnCO-SNAP@P nanoparticles also show a spherical morphology, and the DLS results show that their hydrated particle size is about 168.6nm and the Zeta potential is -18.7mV. The slightly enlarged particle size after drug loading is conducive to the long-term blood circulation of the nanoparticles and the enhanced permeability retention (EPR) effect to passively target the tumor site.

[0077] The responsive release performance of the gas therapeutic agent provided in Example 2 was studied: FL-CO-1 / DAF-FM DA was used as CO and NO fluorescent probes respectively to investigate the production of CO and NO by the gas therapeutic agent in HUVEC and 4T1 cells. Figure 4 As shown in the figure, Figure 4 Fluorescence images of CO and NO after incubation of HUVEC cells with the gas therapeutic agent provided in Example 2 of the present application for different time periods. Figure 5 The CO and NO fluorescence images after the gas therapy agent provided in Example 2 of the present application was incubated with 4T1 cells for different time periods. As can be seen from the figure, almost no green fluorescence was observed in HUVEC cells. This is because the H2O2 / GSH concentration in HUVEC cells is low and CO and NO gases cannot be effectively produced. However, green fluorescence can be observed in 4T1 cells, which shows that the gas therapy agent prompted 4T1 cells to produce CO and NO gases. The above results show that the gas therapy agent responsively produces CO and NO under the stimulation of H2O2 / GSH in tumor cells.

[0078] The cellular phagocytosis and lysosomal escape of gas therapeutic agents were studied: First, rhodamine B (Rhm B) with red fluorescence was encapsulated in PLGA nanoparticles to obtain RhmB@PLGA nanoparticles (RhmB@P NPs), which have similar physical and chemical properties to gas therapeutic agents. Then, the RhmB fluorescence signal after incubation of Rhm B@P nanoparticles with 4T1 cells at different time points was detected by FCM. The results are shown in Figure 2. Figure 6 As shown, Figure 6 This is a graph showing the results of a study on the cellular phagocytosis and lysosomal escape of the gas therapeutic agent provided in an embodiment of the present application. It can be seen from the graph that the Rhm B@P nanoparticles can be completely phagocytosed by 4T1 cells after being co-incubated with 4T1 cells for 4 hours.

[0079] The gas therapeutic agent provided in Example 2 was used to induce DCs maturation and IFN-β secretion: After different nanoparticles (PBS, SNAP@P, MnCO@P, and the gas therapeutic agent provided in Example 2 (denoted as MnCO-SNAP@P)) were co-incubated with 4T1 cells, 4T1 cells were collected and DNA in the cytoplasm was extracted, and genomic DNA (gDNA) and mitochondrial DNA (mtDNA) in the cytoplasm were detected by real-time fluorescence quantitative PCR. The results are shown in Figure 2. Figure 7 As shown, Figure 7 The results of the study on the induction of DCs maturation and IFN-β secretion by gas therapeutic agents provided in Example 2 of the present application can be seen from the figure. Compared with the SNAP@P and MnCO@P groups, a large amount of gDNA and mtDNA were detected in the 4T1 cells treated with the MnCO-SNAP@P group. This result proves that the mitochondria and nuclei of 4T1 cells were damaged after MnCO-SNAP@P treatment, and a large amount of mitochondrial DNA and nuclear DNA were released into the cytoplasm, which can effectively promote DCs maturation and stimulate IFN-β secretion.

[0080] The in vivo anti-tumor effect of the gas therapeutic agent provided in Example 2 was investigated: tumor-bearing mice were treated with different nanoparticles (PBS, PLGA NPs, SNAP@P NPs, MnCO@P NPs, and the gas therapeutic agent provided in Example 2 (denoted as MnCO-SNAP@PNPs)). During the 16-day treatment of the tumor-bearing mice, the changes in the tumor volume of the mice were monitored. The results are as follows: Figure 8 As shown, Figure 8 This is a graph showing the in vivo anti-tumor effect of the gas therapeutic agent provided in Example 2 of the present application. It can be seen from the figure that the tumor volume of the PBS group and the PLGA group has been growing at a relatively high rate, while the volume growth of the MnCO-SNAP@P group was significantly inhibited (e.g. Figure 8In addition, on the 16th day of treatment, the tumor volume inhibition rate of each experimental group was calculated according to the tumor volume of each group, as shown in a. Figure 8 As shown in b, the tumor inhibition rates of the PLGA group, SNAP@P group, MnCO@P group and MnCO-SNAP@P group were 3.91%, 40.97%, 50.88% and 70.95%, respectively.

[0081] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0082] In the present application, in the absence of any contrary description, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the description of the present specification, the terms "including", "comprising", etc. refer to "including but not limited to". In this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of the associated objects, indicating that there may be three relationships, for example, A and / or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A, B can be singular or plural. In this article, "at least one" refers to one or more, and "plural" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of singular or plural items. For example, "at least one of a, b, or c", or "at least one of a, b and c", can both mean: a, b, c, ab (i.e. a and b), ac, bc or abc, where a, b, c can be single or plural, respectively.

[0083] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A multi-responsive nano gas therapeutic agent, characterized in that: The gas therapeutic agent includes PLGA-SNAP polymer-coated MnCO nanoparticles.

2. The multi-responsive nano gas therapeutic agent according to claim 1, characterized in that: In the nanoparticles, the mass ratio of PLGA-SNAP polymer to MnCO is 10:(1.5-3).

3. The multi-responsive nano gas therapeutic agent according to claim 1, characterized in that: The particle size of the nanoparticles is 80-250 nm.

4. A method for preparing a multi-responsive nano gas therapeutic agent, characterized in that: The method comprises: The amino-terminated poly(lactic acid-co-glycolic acid) and S-nitroso-N-acetyl-DL-penicillamine are subjected to an amidation reaction to graft S-nitroso-N-acetyl-DL-penicillamine onto the poly(lactic acid-co-glycolic acid) to obtain a PLGA-SNAP polymer; The PLGA-SNAP polymer is coated on MnCO by an emulsification-solvent evaporation method to obtain a gas therapeutic agent.

5. The method for preparing the multi-responsive nano gas therapeutic agent according to claim 4, characterized in that: The step of subjecting the amino-terminated polylactic acid-glycolic acid copolymer and S-nitroso-N-acetyl-DL-penicillamine to an amidation reaction to graft S-nitroso-N-acetyl-DL-penicillamine onto the polylactic acid-glycolic acid copolymer to obtain a PLGA-SNAP polymer comprises: The amino-terminated polylactic acid-glycolic acid copolymer, a condensation agent, an activator and S-nitroso-N-acetyl-DL-penicillamine are mixed and dissolved in a first solvent, and then an amidation reaction is performed to graft S-nitroso-N-acetyl-DL-penicillamine onto the polylactic acid-glycolic acid copolymer to obtain a PLGA-SNAP polymer.

6. The method for preparing the multi-responsive nano gas therapeutic agent according to claim 5, characterized in that: The condensing agent includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

7. The method for preparing the multi-responsive nano gas therapeutic agent according to claim 5, characterized in that: The activator comprises N-hydroxysuccinimide; and / or The first solvent includes dimethyl sulfoxide.

8. The method for preparing the multi-responsive nano gas therapeutic agent according to claim 4, characterized in that: The method of coating the PLGA-SNAP polymer on MnCO by an emulsification-solvent evaporation method to obtain a gas therapeutic agent comprises: Dissolving the PLGA-SNAP polymer in a second solvent, and then mixing with MnCO to obtain an organic phase; The organic phase and the bovine serum albumin aqueous solution are mixed and ultrasonically emulsified to obtain an emulsion; The emulsion and deionized water are mixed and stirred, and then the organic solvent is evaporated to obtain a gas therapeutic agent.

9. The method for preparing the multi-responsive nano gas therapeutic agent according to claim 8, characterized in that: The second solvent includes dichloromethane.

10. An application of a multi-responsive nano gas therapeutic agent, characterized in that: The application includes using the multi-responsive nano gas therapeutic agent according to any one of claims 1 to 3 or the multi-responsive nano gas therapeutic agent prepared by the method according to any one of claims 4 to 9 for the preparation of drugs.

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