CO hydrogel nano-drug delivery system as well as preparation method and application thereof

Abstract

The invention provides a CO hydrogel nano-drug delivery system, which comprises a nano-core formed by self-assembly of glucose oxidase and human serum albumin, and a first nano-particle formed by entrapping manganese carbonyl into the nano-core through hydrophobic interaction, metal ions are adsorbed on the surfaces of the first nanoparticles through electrostatic interaction to form second nanoparticles, and the second nanoparticles are mixed with polyethylene glycol diacrylate to form a mixture; the mixture is subjected to in-situ polymerization in a physiological environment in which glucose exists to form hydrogel wrapping the second nanoparticles, and the CO hydrogel nano drug delivery system is formed. The nano drug delivery system disclosed by the invention can activate systemic immune response by enhancing immunogen cell death and improving a tumor microenvironment, and meanwhile, GOx and CO can be prevented from leaking by utilizing the protection effect of the in-situ hydrogel, so that the safety of normal tissues is greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a CO hydrogel nano drug delivery system and a preparation method and application thereof. Background Art

[0002] Cancer is also called malignant tumor. Cancer is a disease caused by the loss of normal regulation of body cells and excessive proliferation. Excessively proliferating cells are called cancer cells. Cancer cells can often invade surrounding tissues and even metastasize to other parts of the body through the body's circulatory system and / or lymphatic system. There are many types of cancer, and the severity of the disease depends on the location of the cancer cells, the degree of malignancy, and whether metastasis occurs. How to effectively treat cancer is a difficult problem in the current medical field.

[0003] In situ tumor vaccines are an emerging tumor immunotherapy method that releases vaccine components directly at the tumor resection site or inside the tumor through local administration to activate the body's immune response and prevent tumor recurrence and metastasis. However, in situ tumor vaccines still face some challenges, such as insufficient tumor antigens released by tumor cells, which limits the uptake of antigens by immune cells; insufficient processing of antigens by antigen-presenting cells, which limits the generation of antigen-specific T cell responses; and the immunosuppressive microenvironment of the tumor, which leads to the exhaustion and death of effector cells.

[0004] Currently, more and more researchers are using delivery technology and designing different types of delivery formulations to enhance the therapeutic effect of tumor vaccines, such as delivery of chemotherapy drugs and biomolecule delivery. By delivering chemotherapy drugs, tumor cell death can be induced and tumor antigens can be released. These antigens can be recognized by the immune system. However, chemotherapy drugs are toxic to normal cells and may cause systemic toxicity. In addition, due to the high osmotic pressure inside the tumor, the drug cannot enter the tumor to improve the immunosuppressive microenvironment. By delivering biomolecules, such as oncolytic viruses and cytolytic peptides, biomacromolecules can directly destroy tumor cell membranes or regulate cell death, thereby releasing tumor antigens. However, the large-scale production and purification of biomolecules may be complex and costly, and some biomolecules may cause adverse reactions or immunosuppression. Moreover, these methods still cannot overcome the problem of preventing the body from producing a strong anti-tumor immune response due to insufficient antigen release, poor adjuvant effect, and immunosuppression in the tumor microenvironment.

[0005] Therefore, there is an urgent need to develop an in situ tumor vaccine that can overcome the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a CO hydrogel nano drug delivery system and a preparation method thereof in view of the deficiencies of the prior art, which can activate systemic immune response by enhancing immunogenic cell death and improving the tumor microenvironment. At the same time, the protective effect of in situ hydrogel can prevent GOx and CO from leaking, greatly increasing the safety of normal tissues.

[0007] According to a first aspect of the present invention, a CO hydrogel nano drug delivery system is provided, comprising a nano core formed by self-assembly of glucose oxidase and human albumin, carbonyl manganese is encapsulated into the nano core through hydrophobic interaction to form a first nanoparticle, metal ions are adsorbed on the surface of the first nanoparticle through electrostatic interaction to form a second nanoparticle, and the second nanoparticle is mixed with polyethylene glycol diacrylate to form a mixture;

[0008] The mixture is polymerized in situ in a physiological environment where glucose exists to form a hydrogel that encapsulates the second nanoparticles, thereby forming a CO hydrogel nano drug delivery system.

[0009] As an optional embodiment, the Zeta potential of the first nanoparticles is -20mV to -25mV, the particle size of the second nanoparticles is 175nm to 180nm, and the Zeta potential is 5mV to 10mV.

[0010] As an optional embodiment, in the second nanoparticles, the mass proportion of glucose oxidase is 10% to 20%, and the mass proportion of carbonyl manganese compound is 10% to 25%.

[0011] As an optional embodiment, the metal ion is a metal ion that can undergo a Fenton reaction with hydrogen peroxide.

[0012] According to a second aspect of the present invention, a method for preparing the aforementioned CO hydrogel nano drug delivery system is provided, comprising the following steps:

[0013] S1. adding human albumin and glucose oxidase into deionized water respectively, mixing, heating and stirring, so that the peptide chains of human albumin and glucose oxidase are fully unfolded;

[0014] S2, slowly adding the carbonyl manganese solution to the solution obtained in step S1 under heating and stirring conditions, transferring the solution to an ice bath and stirring immediately after the addition, and encapsulating the carbonyl manganese in the nanocore formed by self-assembly of glucose oxidase and human albumin through hydrophobic interaction to obtain a first nanoparticle solution;

[0015] S3, adding the aqueous solution of the metal ion salt to the first nanoparticle solution obtained in step S2, stirring under nitrogen protection, adsorbing the metal ions on the surface of the first nanoparticles through electrostatic interaction, and continuing to remove residual small molecules and excess deionized water by ultrafiltration under nitrogen protection to form a second nanoparticle solution;

[0016] S4. Add polyethylene glycol diacrylate to the second nanoparticle solution obtained in step S3 to obtain a mixture, and the mixture is in situ polymerized in a physiological environment in the presence of glucose to form a hydrogel encapsulating the second nanoparticles, thereby forming a CO hydrogel nano drug delivery system.

[0017] As an optional embodiment, in a mixed solution of human albumin and glucose oxidase, the concentration of glucose oxidase is 0.2 mg / mL to 0.3 mg / mL, the concentration of human albumin is 1.5 mg / mL to 1.7 mg / mL, the concentration of carbonyl manganese solution is 2 mg / mL to 3 mg / mL, and the mass ratio of carbonyl manganese to glucose oxidase is (5:4) to (7:4).

[0018] As an optional embodiment, the metal ion salt includes chlorophyll tetrahydrate, the concentration of the aqueous solution of the metal ion salt is 3 mg / mL to 5 mg / mL, and the mass ratio of carbonyl manganese to the metal ion salt is (9:4) to (11:4).

[0019] As an optional embodiment, the concentration of polyethylene glycol diacrylate is 20% w / v, and the mass ratio of the second nanoparticles to the polyethylene glycol diacrylate is (1:6) to (1:10).

[0020] According to a third aspect of the present invention, there is provided a use of the aforementioned CO hydrogel nano drug delivery system in the preparation of tumor immunotherapy drugs.

[0021] According to a fourth aspect of the present invention, a CO hydrogel gas in situ tumor vaccine is provided, comprising the aforementioned CO hydrogel nano drug delivery system.

[0022] It can be seen from the above technical scheme of the present invention that the CO hydrogel nano drug delivery system proposed by the present invention is a new type of CO hydrogel gas in situ tumor vaccine, which uses human serum albumin and glucose oxidase as carriers, utilizes hydrophobic interaction to encapsulate carbonyl manganese compounds to form nanoparticles, albumin is negatively charged, and electrostatic interaction is utilized to adsorb metal ions on the surface of nanoparticles, and polyethylene glycol diacrylate is added to the nanoparticles;

[0023] In situ injection into the tumor site, due to the presence of glucose in the tumor site, it reacts with glucose oxidase to generate hydrogen peroxide, promoting the release of CO, and the Fenton reaction to generate ·OH to induce PEGDA polymerization to form a hydrogel to encapsulate the nanoparticles, avoiding the toxicity of the drug to normal tissues and protecting the nanoparticles from degradation. At the same time, it allows the loaded drug to be released precisely in time and space, enhancing the immune response of the tumor vaccine;

[0024] The ·OH generated by the Fenton reaction not only induces the in situ gelation of PEGDA, but also effectively amplifies immunogenic cell death due to the large amount of reactive oxygen species produced in the cells;

[0025] At the same time, the release of CO induces the production of intracellular ROS, which promotes the maturation of DC cells by enhancing immunogenic cell death, releasing tumor-associated antigens (TAA) and damage-associated molecular patterns (DAMPs), and the maturation of DC cells will assist TAA in promoting T cells to enhance lymphocyte infiltration and activate systemic anti-tumor immune response. At the same time, CO regulates excessive inflammatory factors in the tumor site, reduces the infiltration ratio of MDSC in the tumor site, polarizes M2 macrophages to M1 macrophages, thereby reducing the expression of immunosuppressive factors such as TGF-β and IL-10 in the microenvironment, and further relieves the inhibition of DC cell maturation, improves the immunosuppressive microenvironment, induces effective in situ gas vaccination and initiates effective anti-tumor immune response, and enhances the effect of vaccine.

[0026] In this way, CO gas therapy, starvation therapy and chemodynamic therapy are used synergistically and injected in situ into the tumor to increase immunogenic cell death, enhance tumor immunotherapy, and have a strong vaccine effect.

[0027] The CO hydrogel nano drug delivery system of the present invention has the advantages of maintaining drug activity, strong drug efficacy and low incidence of side effects. At the same time, carrier materials such as human albumin and polyethylene glycol diacrylate have good biocompatibility and degradability, reducing their toxicity in the body, which is an important prerequisite for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the synthesis route of the CO hydrogel nano drug delivery system of the present invention.

[0029] Figure 2 It is a particle size characterization diagram of HGM-Fe in the example of the present invention.

[0030] Figure 3 It is a potential characterization diagram before and after ferrous metal ion loading in the example of the present invention.

[0031] Figure 4It is a hydrogel formation diagram in an example of the present invention; wherein 4a is when there is no glucose, and 4b is when there is glucose.

[0032] Figure 5 1 and 2 are transmission / scanning electron micrographs of the examples of the present invention, respectively showing a transmission electron micrograph of HGM-Fe nanoparticles and a scanning electron micrograph of HGM-Fe / PEGDA hydrogel.

[0033] Figure 6 The anti-tumor effect of CO hydrogel as a gas vaccine in tumor-bearing mice in the example of the present invention is shown in Figure 3, which are the experimental group, the control group and the normal saline group.

[0034] Figure 7 It is a flow cytometric diagram of MDSCs in tumor tissue in the example of the present invention.

[0035] Figure 8 This is the vaccine effect of CO hydrogel as a gas vaccine in mice in the example of the present invention. DETAILED DESCRIPTION

[0036] In order to better understand the technical content of the present invention, specific embodiments are given and described as follows in conjunction with the accompanying drawings.

[0037] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to include all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways.

[0038] Tumor in situ vaccines provide a new approach for personalized immunotherapy against various tumors. They kill established tumors by inducing immunogenic cell death, leading to the release of tumor antigens and initiating systemic anti-tumor immune responses. However, because tumors construct a suppressive immune microenvironment within them, they induce the infiltration of suppressive immune cells, resulting in insufficient immunogenicity. In addition, the immunosuppressive microenvironment limits the effectiveness of effector T cells and promotes tumor immune escape.

[0039] Carbon monoxide gas has a high affinity for hemoglobin (Hb) and has side effects such as low bioavailability. Injection through the tail vein may cause CO to combine with Hb in the blood and cause poisoning. In addition, low bioavailability leads to less CO content in the tumor site, and the expected therapeutic effect cannot be achieved.

[0040] Therefore, the present invention constructs a CO hydrogel nano drug delivery system, adopts CO hydrogel as a gas in situ vaccine, forms a hydrogel at the tumor site, protects the nanoparticles from degradation, and allows the loaded drug to be released precisely in situ in time and space, and synergistically enhances immunogenic cell death through the Fenton reaction and the released CO, and improves the tumor microenvironment to activate the systemic immune response, providing a promising treatment strategy.

[0041] CO hydrogel nano drug delivery system

[0042] Combination Figure 1 As shown, in an exemplary embodiment of the present invention, a CO hydrogel nano drug delivery system is provided, comprising a nanocore formed by self-assembly of glucose oxidase (GOx) and human albumin (HSA), carbonyl manganese (MnCO) is encapsulated into the nanocore through hydrophobic interaction to form a first nanoparticle HSA-GOx-MnCO, metal ions are adsorbed on the surface of the first nanoparticle through electrostatic interaction to form a second nanoparticle HGM-M, and the second nanoparticle is mixed with polyethylene glycol diacrylate (PEGDA) to form a mixture HGM-M / PEGDA.

[0043] In the physiological environment of glucose, the mixture HGM-M / PEGDA reacts with GOx to generate hydrogen peroxide, which promotes the metal ion-mediated Fenton reaction and produces ·OH, thereby initiating the in situ polymerization of PEGDA to form a hydrogel that wraps the second nanoparticles, forming a CO hydrogel nano-drug delivery system.

[0044] In an optional example, the Zeta potential of the first nanoparticle HSA-GOx-MnCO is -20 to -25 mV.

[0045] In an optional example, the particle size of the second nanoparticle HGM-M is 175-180 nm, and the Zeta potential is 5-10 mV.

[0046] In an optional example, in the second nanoparticle HGM-M, the mass proportion of glucose oxidase is 10% to 20%, and particularly preferably 11.7% to 17.6%; the mass proportion of carbonyl manganese compound is 10% to 25%, and particularly preferably 14.6% to 21.9%.

[0047] In an optional example, the metal ion is a metal ion that can undergo a Fenton reaction with hydrogen peroxide, including a ferrous ion.

[0048] Preparation method

[0049] In another exemplary embodiment of the present invention, a method for preparing the aforementioned CO hydrogel nano drug delivery system is provided, comprising the following steps:

[0050] S1. adding human albumin and glucose oxidase into deionized water respectively, mixing, heating and stirring, so that the peptide chains of human albumin and glucose oxidase are fully unfolded;

[0051] S2, slowly adding the carbonyl manganese solution to the solution obtained in step S1 under heating and stirring conditions, transferring the solution to an ice bath and stirring immediately after the addition, and encapsulating the carbonyl manganese in the nanocore formed by self-assembly of glucose oxidase and human albumin through hydrophobic interaction to obtain a first nanoparticle solution (HSA-GOx-MnCO);

[0052] S3, adding the aqueous solution of metal ion salt to the first nanoparticle solution obtained in step S2, stirring under nitrogen protection, adsorbing the metal ions on the surface of the first nanoparticles through electrostatic interaction, and continuing to remove residual small molecules and excess deionized water by ultrafiltration under nitrogen protection to form a second nanoparticle solution (HGM-M);

[0053] S4. Add polyethylene glycol diacrylate to the second nanoparticle solution obtained in step S3 to obtain a mixture (HGM-M / PEGDA), and polymerize the mixture in situ in a physiological environment in the presence of glucose to form a hydrogel encapsulating the second nanoparticles, thereby forming a CO hydrogel nano drug delivery system.

[0054] In an optional example, in the mixed solution of human serum albumin and glucose oxidase, the concentration of glucose oxidase is 0.2 mg / mL to 0.3 mg / mL, and the concentration of human serum albumin is 1.5 mg / mL to 1.7 mg / mL.

[0055] In an optional example, in step S1, the temperature of heating and stirring is 64±1°C to fully unfold the peptide chain.

[0056] In an optional example, in step S2, the concentration of the carbonyl manganese solution is 2 mg / mL to 3 mg / mL, and the mass ratio of carbonyl manganese to glucose oxidase is (5:4) to (7:4), and is particularly preferably 3:2.

[0057] In an optional example, in step S3, the metal ion salt includes tetrahydrated manganese chloride, the concentration of the aqueous solution of the metal ion salt is 3 mg / mL to 5 mg / mL, and the mass ratio of carbonyl manganese to the metal ion salt is (9:4) to (11:4), and is particularly preferably 5:2.

[0058] In an optional example, in step S4, the concentration of polyethylene glycol diacrylate is 20% w / v, and the mass ratio of the second nanoparticles to the polyethylene glycol diacrylate is (1:6) to (1:10), and is particularly preferably 1:8.

[0059] It is understood that the solvent of polyethylene glycol diacrylate is deionized water.

[0060] In another exemplary embodiment, an aqueous solution of glucose (at a concentration of 0.9 mg / mL) is added to the mixture HGM-M / PEGDA obtained in step S4, and after gentle shaking, it can be polymerized in situ within a few minutes to form a hydrogel; thus, it can be proven that the drug delivery system of the present invention can gel in situ in vivo.

[0061] In order to observe whether the gelation is formed, the hydrogels in vitro of the present invention are all prepared by adding glucose to HGM-M / PEGDA.

[0062] In other embodiments of the present invention, there is also provided a use of the aforementioned CO hydrogel nano drug delivery system in the preparation of tumor immunotherapy drugs.

[0063] In a specific embodiment, a CO hydrogel gas in situ tumor vaccine comprising the aforementioned CO hydrogel nano drug delivery system is provided, wherein the gas in situ tumor vaccine is delivered via Fe 2+ The mediated Fenton reaction and the released CO synergistically enhance immunogenic cell death and promote antigen presentation; in addition, CO can significantly reduce the activation and expansion of MDSC cells, reduce the infiltration ratio of MDSC in the tumor site, polarize M2 macrophages to M1 type, and reprogram myeloid-derived suppressor cells, thereby reducing the expression of immunosuppressive factors in the microenvironment, improving the immunosuppressive microenvironment, inducing effective in situ gas vaccination and initiating effective anti-tumor immune response.

[0064] For better understanding, the present invention is further described below in conjunction with several specific examples, but the preparation process is not limited thereto, and the content of the present invention is not limited thereto.

[0065] Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0066] The human albumin solution used in the following examples of the present invention was purchased from Octa Pharma, the carbonyl manganese compound was purchased from Shanghai Huawei Raycus, polyethylene glycol diacrylate was purchased from Anage Chemical Co., Ltd., and ferrous chloride tetrahydrate was purchased from Shanghai Macklin.

[0067] Example 1

[0068] (1) Take 19 mL of deionized water and heat it to 64°C in a water bath, add 1 mL of glucose oxidase (6 mg / mL) thereto, draw 0.16 mL of 0.2 g / mL human albumin with a 1 mL syringe and slowly add it thereto, stir at 500 rpm for 5 min under the condition of heating in a water bath to fully unfold the peptide chain, weigh 4 mg of carbonyl manganese compound with an electronic balance and dissolve it in 2 mL of ethanol, slowly adjust the speed to 1200 rpm for high-speed stirring, slowly add the carbonyl manganese solution dropwise with a 2.5 mL syringe within 1 min, quickly transfer to ice bath conditions, and stir at 500 rpm for 10 min to form HGM.

[0069] (2) Weigh ferrous chloride tetrahydrate, add water to dissolve it so that the concentration of ferrous chloride tetrahydrate is 4 mg / mL, take 800 μL of ferrous chloride tetrahydrate solution and add it to the HGM solution obtained in step (1), stir at room temperature for 10 min under nitrogen protection, and then, ultrafilter and concentrate the solution to about 3 mL through a 30 kDa ultrafiltration membrane under nitrogen protection to remove excess water and residual ions to form HGM-Fe nanoparticles.

[0070] (3) Take 400 μL of HGM-Fe solution and add 100 μL of 20% (w / v) PEGDA to obtain a mixture of HGM-Fe / PEGDA.

[0071] Example 2

[0072] [Particle size test]

[0073] The HGM-Fe nanoparticles prepared according to the method in Example 1 were measured by a Malvern particle size analyzer. The particle size of the HGM-Fe nanoparticles is as follows: Figure 2 shown.

[0074] It can be seen from the figure that the hydrated particle size of HGM-Fe nanoparticles is 175nm~180nm, and the particle size is uniform and stable.

[0075] Example 3

[0076] [Potential test]

[0077] The HGM and HGM-Fe nanoparticles prepared according to the method in Example 1 were tested for their potential using a Malvern particle size analyzer. The potential results of the two nanoparticles are shown in Figure 3 shown.

[0078] As can be seen from the figure, the potential of the nanoparticles HGM without metal ions is -20mV to -25mV; the potential of the nanoparticles HGM-Fe with ferrous chloride tetrahydrate is 5mV to 10mV; this indicates that the metal Fe 2+The ions were successfully loaded on the HGM surface.

[0079] Example 4

[0080] [Gel forming test]

[0081] 0.9 mg / mL glucose solution was added to the mixture HGM-Fe / PEGDA prepared according to the method in Example 1, and the mixture was gently shaken to form a hydrogel. The hydrogel formation result is shown in FIG. Figure 4 shown.

[0082] As can be seen from the figure, in the absence of glucose, it appears liquid ( Figure 4 a), after adding glucose solution, GOx releases hydrogen peroxide, mediates CO release and Fenton reaction, generates free radicals to induce PEGDA polymerization to form hydrogel ( Figure 4 b).

[0083] Example 5

[0084] [Morphology test]

[0085] The HGM-Fe nanoparticles prepared according to the method in Example 1 and the hydrogel formed by adding 0.9 mg / mL glucose solution to HGM-M / PEGDA were observed under transmission / scanning electron microscope for the morphology of the nanoparticles HGM-Fe and the hydrogel. The electron microscopy results of the two nanoparticles are as follows: Figure 5 shown.

[0086] As can be seen from the figure, HGM-Fe nanoparticles show a uniform spherical structure under transmission electron microscopy. At the same time, the particle size of the nanoparticles observed in the figure is basically consistent with the particle size of the nanoparticles measured by the particle size analyzer, which meets the standards of nanoparticles. The hydrogel HGM-Fe / PEGDA was observed to have a network structure with uniform pores under a scanning electron microscope, indicating that the hydrogel was successfully prepared.

[0087] Example 6

[0088] [Determination of component content]

[0089] The content of GOx in HGM-Fe was determined by a GOx assay kit, and the content of carbonyl manganese was measured by an ultraviolet spectrophotometer at 340 nm.

[0090] The test shows that in 30 mg of HGM-Fe, the content of glucose oxidase is 3.5 mg to 5.2 mg, and the content of carbonyl manganese compound is 4.4 mg to 6.5 mg. The mass proportion of glucose oxidase is 11.7% to 17.6%, and the mass proportion of carbonyl manganese is 14.6% to 21.9%.

[0091] The HG-Fe / PEGDA, HGM / PEGDA, and HGM-Fe / PEGDA used in the following examples are:

[0092] HG-Fe / PEGDA was prepared according to the method of Example 1, wherein carbonyl manganese was not added in step (1);

[0093] HGM / PEGDA is a mixture of HGM prepared according to the method of Example 1 and 20% (w / v) PEGDA (the mass ratio of HGM to PEGDA is 1:8);

[0094] HGM-Fe / PEGDA was prepared according to the method of Example 1.

[0095] Example 7

[0096] [Antitumor effect]

[0097] (1) Select tumors with a volume of 50-80 mm 3 4T1 mice were randomly divided into four groups, 6 in each group. They were normal saline group; human albumin-glucose oxidase-ferrous ion-polyethylene glycol diacrylate group (HG-Fe / PEGDA); human albumin-glucose oxidase-carbonyl manganese compound-polyethylene glycol diacrylate group (HGM / PEGDA); human albumin-glucose oxidase-carbonyl manganese compound-ferrous ion-polyethylene glycol diacrylate group (HGM-Fe / PEGDA). The above four groups were injected intratumorally, and the drug was injected orthotopically into the tumor site of the mouse on the first day.

[0098] (2) The tumor size was recorded continuously from the first day after administration, and the tumor volume was calculated according to the following: width*width*length / 2. The mice were sacrificed two weeks later.

[0099] (3) The results are as follows Figure 6 As shown, the efficacy of antitumor therapy was evaluated in 4T1 breast cancer tumor-bearing mice by different treatments.

[0100] The tumor in the normal saline group grew very fast and was ineffective. The treatment effects of the HG-Fe / PEGDA and HGM / PEGDA groups were not very ideal, while the HGM-Fe / PEGDA group showed very good anti-tumor effects. HG-Fe / PEGDA did not encapsulate carbonyl manganese, and only had a Fenton reaction mediated by ferrous ions at the tumor site, without CO gas release, and could not have a vaccine effect. HGM / PEGDA did not have a Fenton reaction mediated by ferrous ions, and could not produce hydroxyl radicals well, and could not mediate PEGDA polymerization to form a hydrogel, affecting the effect of inducing immunogenic death.

[0101] Therefore, it can be proved that the HGM-Fe / PEGDA of the present invention has a better anti-tumor effect by using CO gas therapy in synergy with starvation therapy and chemodynamic therapy.

[0102] Example 8

[0103] [Improving the tumor microenvironment]

[0104] Tumor cells can directly activate myeloid suppressor cells (MDSCs) to escape immune surveillance. Therefore, flow cytometry was used to evaluate the suppressive effect of MDSCs.

[0105] Select tumors with a volume of 50-80 mm 3 4T1 mice were randomly divided into four groups, 6 mice in each group, namely normal saline group; human albumin-glucose oxidase-ferrous ion-polyethylene glycol diacrylate group (HG-Fe / PEGDA); human albumin-glucose oxidase-carbonyl manganese compound-polyethylene glycol diacrylate group (HGM / PEGDA); human albumin-glucose oxidase-carbonyl manganese compound-ferrous ion-polyethylene glycol diacrylate group (HGM-Fe / PEGDA).

[0106] On the fifth day after administration, mice were dissected, lymphatic tissues and tumor tissues of tumor-bearing mice were collected, 500 μL of culture medium was added and the tissues were chopped, and 500 μL of enzyme lysis solution prepared with collagenase type I, collagenase type IV and hyaluronidase in a ratio of 1:1:1 was added, and the cells were shaken and incubated at 37°C for 60 minutes. After the incubation, the cells were passed through a 70 μm sieve, and the single-cell suspension of tumors was collected by adding PBS, centrifuged at 2000 rpm for 5 minutes, and the cells were collected after sieving again.

[0107] Antibodies CD11b-FITC and Cy-6C / 6G-APC were used to label MDSCs cells in tumor tissues, which were then washed and resuspended for flow cytometry detection. The results were as follows: Figure 7 shown.

[0108] As can be seen from the figure, the proportion of Cr-1+CD11b+ in HG-Fe / PEGDA, HGM / PEGDA, and HGM-Fe / PEGDA was lower than that in the normal saline group, especially in the HGM-Fe / PEGDA treatment group, which was 53.59%, which was significantly lower than 79.94% in the control group.

[0109] Therefore, it can be demonstrated that the HGM-Fe / PEGDA of the present invention can reverse the immunosuppressive tumor microenvironment by reducing the expression of MDSCs, thereby inducing effective in situ gas vaccination and initiating an effective anti-tumor immune response.

[0110] Example 9

[0111] [Vaccine effect]

[0112] (1) 4T1 cells were plated in a cell culture flask. After the cells were fully grown, they were treated with different drugs, including normal saline, human albumin-glucose oxidase-ferrous ion-polyethylene glycol diacrylate group (HG-Fe / PEGDA) and human albumin-glucose oxidase-carbonyl manganese compound-ferrous ion-polyethylene glycol diacrylate group (HGM-Fe / PEGDA). The treated 4T1 tumor cells were subcutaneously injected into the left side of immunocompetent BALB / c mice. Ten days later, the surviving 4T1 cells were inoculated again into the right side of the mice.

[0113] (2) The tumor size was recorded continuously from the first day after the injection of right tumor cells, and the tumor volume was calculated according to the following: width*width*length / 2. The mice were killed two weeks later.

[0114] (3) The results are as follows Figure 8 As shown, the efficacy of the vaccine was evaluated in 4T1 breast cancer tumor-bearing mice by different treatments.

[0115] The tumors in the saline group grew very fast and had no effect, the treatment effect in the HG-Fe / PEGDA group was not very ideal, and the tumor growth of mice in the HGM-Fe / PEGDA group was significantly inhibited.

[0116] The results show that the HGM-Fe / PEGDA hydrogel of the present invention can make tumor cells more immunogenic and produce a stronger vaccine effect in systemic anti-tumor immunity.

[0117] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the definition of the claims.

Claims

1. A CO hydrogel nano drug delivery system, characterized in that: The method comprises utilizing a nano core formed by self-assembly of glucose oxidase and human albumin, encapsulating carbonyl manganese into the nano core through hydrophobic interaction to form a first nanoparticle, adsorbing metal ions on the surface of the first nanoparticle through electrostatic interaction to form a second nanoparticle, and mixing the second nanoparticle with polyethylene glycol diacrylate to form a mixture; The mixture is polymerized in situ in a physiological environment where glucose exists to form a hydrogel that encapsulates the second nanoparticles, thereby forming a CO hydrogel nano drug delivery system.

2. The CO hydrogel nano drug delivery system according to claim 1, characterized in that: The Zeta potential of the first nanoparticles is -20 mV to -25 mV, the particle size of the second nanoparticles is 175 nm to 180 nm, and the Zeta potential is 5 mV to 10 mV.

3. The CO hydrogel nano drug delivery system according to claim 1, characterized in that: In the second nanoparticles, the mass proportion of glucose oxidase is 10% to 20%, and the mass proportion of carbonyl manganese compound is 10% to 25%.

4. The CO hydrogel nano drug delivery system according to claim 1, characterized in that: The metal ion is a metal ion that can undergo Fenton reaction with hydrogen peroxide.

5. A method for preparing the CO hydrogel nano drug delivery system according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. adding human albumin and glucose oxidase into deionized water respectively, mixing, heating and stirring, so that the peptide chains of human albumin and glucose oxidase are fully unfolded; S2, slowly adding the carbonyl manganese solution to the solution obtained in step S1 under heating and stirring conditions, transferring the solution to an ice bath and stirring immediately after the addition, and encapsulating the carbonyl manganese in the nanocore formed by self-assembly of glucose oxidase and human albumin through hydrophobic interaction to obtain a first nanoparticle solution; S3, adding the aqueous solution of the metal ion salt to the first nanoparticle solution obtained in step S2, stirring under nitrogen protection, adsorbing the metal ions on the surface of the first nanoparticles through electrostatic interaction, and continuing to remove residual small molecules and excess deionized water by ultrafiltration under nitrogen protection to form a second nanoparticle solution; S4. Add polyethylene glycol diacrylate to the second nanoparticle solution obtained in step S3 to obtain a mixture, and the mixture is in situ polymerized in a physiological environment in the presence of glucose to form a hydrogel encapsulating the second nanoparticles, thereby forming a CO hydrogel nano drug delivery system.

6. The preparation method according to claim 5, characterized in that: In a mixed solution of human albumin and glucose oxidase, the concentration of glucose oxidase is 0.2 mg / mL to 0.3 mg / mL, the concentration of human albumin is 1.5 mg / mL to 1.7 mg / mL, the concentration of carbonyl manganese solution is 2 mg / mL to 3 mg / mL, and the mass ratio of carbonyl manganese to glucose oxidase is (5:4) to (7:4).

7. The preparation method according to claim 5, characterized in that: The metal ion salt includes tetrahydrated manganese chloride, the concentration of the aqueous solution of the metal ion salt is 3 mg / mL to 5 mg / mL, and the mass ratio of carbonyl manganese to the metal ion salt is (9:4) to (11:4).

8. The preparation method according to claim 5, characterized in that: The concentration of polyethylene glycol diacrylate is 20% w / v, and the mass ratio of the second nanoparticles to the polyethylene glycol diacrylate is (1:6) to (1:10).

9. Use of the CO hydrogel nano drug delivery system according to any one of claims 1 to 4 in the preparation of tumor immunotherapy drugs.

10. A CO hydrogel gas in situ tumor vaccine, characterized in that: A CO hydrogel nano drug delivery system comprising any one of claims 1 to 4.

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