Preparation of pyroptosis inducer hemin nano-micelle and application of pyroptosis inducer hemin nano-micelle in tumor immunotherapy

Abstract

The invention discloses preparation of a pyroptosis inducer chlorhematin nano-micelle and application of the pyroptosis inducer chlorhematin nano-micelle in tumor immunotherapy. The preparation method of the chlorhematin nano-micelle comprises the following steps: (1) mixing sodium hyaluronate, NHS (N-hydroxysuccinimide), EDCI (1, 2 '-EDCI) and 2, 2'-dithiodiethylamine dihydrochloride, and reacting to obtain HA-SS-NH2; (2) hemin, NHS and EDCI are subjected to a reaction, a Hemin solution is obtained, then HA-SS-NH2 is dropwise added into the Hemin solution, and a HA-SS-Hemin polymer is obtained through a reaction; and (3) dissolving the HA-SS-Hemin polymer into dimethyl sulfoxide, then dropwise adding into reverse-phase solvent water, and reacting to obtain the chlorhematin nano-micelle. The chlorhematin nano-micelle prepared in the invention has the advantages of uniform size and good stability, can induce pyroptosis of tumor cells, and provides a new strategy for tumor immunotherapy.

Description

Technical Field

[0001] The invention belongs to the field of nano-biomedicine, and particularly relates to the preparation of hemin nano-micelles, a cell pyroptosis inducer, and the application of the nano-micelles in tumor immunotherapy. Background Art

[0002] According to the latest "Global Cancer Data" released by the International Agency for Research on Cancer (IARC), in 2022 alone, there will be 20 million new cancer cases and 9.7 million deaths worldwide. The incidence and mortality of cancer worldwide are on the rise year by year, seriously threatening people's quality of life and health. Traditional clinical treatments for malignant tumors mainly include surgery, radiotherapy and chemotherapy, but these three major tumor treatment technologies still face their own limitations. Furthermore, the latest data show that the rapid growth in the number of new cases of breast cancer (2.3 million new cases in 2022 alone, 11.6%) has become the second largest cause of cancer incidence worldwide, seriously endangering the lives of women. Among them, triple-negative breast cancer (TNBC) refers specifically to a type of breast cancer in which estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor-2 (HER-2) are all negative, with the characteristics of strong invasiveness, rapid progression, easy recurrence, easy metastasis and poor prognosis. At present, the main clinical treatment options for breast cancer are still surgery and chemoradiotherapy, which also include adjuvant hormone endocrine therapy and HER-2 targeted therapy, such as trastuzumab and pertuzumab. However, TNBC cannot be treated with endocrine therapy because it does not express ER and PR receptors. It also cannot benefit from HER-2-related antibody targeted therapy due to the lack of HER-2 receptors. Traditional therapies are even more difficult to eradicate TNBC primary lesions, and the risk of recurrence is high. Therefore, TNBC has become the most difficult type of breast cancer to treat, so it is of great significance to develop effective treatment strategies for TNBC.

[0003] Tumor immunotherapy has attracted much attention due to its significant clinical efficacy. However, due to differences in the tumor's own immune response, "cold tumors" with low immunogenicity respond poorly to immunotherapy, limiting the development of immunotherapy. Reprogramming the tumor immune microenvironment and converting "cold tumors" into "hot tumors" is an important means to improve the responsiveness of tumor immunotherapy. Pyroptosis, as a new type of programmed death, can cause a strong inflammatory response by releasing proinflammatory cytokines and danger signal molecules (DAMPs), thereby promoting the activation and infiltration of immune cells and activating the immune system. It plays an important role in anti-tumor immune response, and pyroptosis has gradually become an emerging strategy in tumor immunotherapy. However, many traditional pyroptosis inducers, including some specific small molecules, ions such as Ca 2+Or chemotherapy drugs such as doxorubicin (DOX), decitabine (DAC), methotrexate, metformin, RIG-1 agonists, etc., have problems such as rapid clearance from the blood circulation, obvious drug resistance, non-specific biodistribution, poor clinical effects of conventional chemotherapy, and adverse reactions caused by high-dose drugs. Therefore, it is challenging to induce pyroptosis in specific tumor cells, and how to improve the tumor targeting and responsiveness of pyroptosis inducers is an important scientific issue in this field. Nanomedicines have the advantages of reduced toxic side effects, targeted delivery, and responsive release, which bring hope to solving the problems of clinical drug treatment, but they also face the challenge of biosafety. Therefore, it is urgent to develop safe and effective new nano-pyroptosis-inducing drugs to drive tumor immunotherapy.

[0004] Hemin is a Toll-like receptor 4 agonist derived from the body with pro-inflammatory and pro-oxidative functions. It has a stable chemical structure and is easy to modify. It is considered an ideal iron supplement in the clinic. It is used as a raw material in the pharmaceutical industry to develop anti-tumor and anti-anemia drugs, and is increasingly favored by people. Monomeric hemin exerts anti-tumor effects due to its potential toxicity. However, as a highly hydrophobic small molecule compound, hemin has low bioavailability and obvious drug delivery problems. At present, the hemin molecule is modified through nanotechnology to improve its targeted delivery problem, so that it can be released in response to the tumor microenvironment, and used to develop drugs that induce tumor cell pyroptosis to achieve tumor immunotherapy. This has not been reported in domestic and foreign research. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing hemin nano-micelles.

[0006] Another object of the present invention is to provide hemin nano-micelles prepared by the method.

[0007] Another object of the present invention is to provide an application of the hemin nano-micelles.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A method for preparing hemin nano-micelles comprises the following steps:

[0010] (1) Synthesis of HA-SS-NH2:

[0011] Sodium hyaluronate is dissolved in a solvent, and then N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) are added, and after stirring for reaction, 2,2'-dithiodiethylamine dihydrochloride is added, and the stirring reaction is continued. After the reaction is completed, dialyzation is performed, and freeze-drying is performed to obtain HA-SS-NH2 powder;

[0012] (2) Synthesis of HA-SS-Hemin polymer:

[0013] Dissolve hemin in a solvent, then add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and stir to react, to obtain a Hemin solution; dissolve the HA-SS-NH2 powder obtained in step (1) in water to obtain a HA-SS-NH2 solution; then slowly drop the HA-SS-NH2 solution into the Hemin solution, mix well, and continue to stir to react, after the reaction is completed, dialyze, concentrate, and freeze-dry to obtain a HA-SS-Hemin polymer;

[0014] (3) Synthesis of Hemin Nanomicelles:

[0015] The HA-SS-Hemin polymer obtained in step (2) is dissolved in dimethyl sulfoxide (DMSO) to obtain a HA-SS-Hemin polymer solution; then the HA-SS-Hemin polymer solution is added dropwise to continuously stirred water (as a reverse phase solvent), stirred for reaction, dialyzed, concentrated, and filtered to obtain hemin nanoemulsification.

[0016] The weight average molecular weight (Mw) of the sodium hyaluronate described in step (1) is preferably 4980.

[0017] The molar ratio of sodium hyaluronate, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2,2'-dithiodiethylamine dihydrochloride described in step (1) is 1:36-60:36-60:36-60; preferably 1:36:36:36 (Note: there are 12 monomer molecules in hyaluronic acid with a molecular weight of 4980, that is, 12 carboxyl groups are activated for subsequent amide reaction, that is, the molar ratio of carboxyl groups, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2,2'-dithiodiethylamine dihydrochloride in sodium hyaluronate (i.e., the ratio of chemical reaction equivalents) is 1:3-5:3-5, 3-5; preferably 1:3:3:3).

[0018] The solvent described in step (1) is a solvent obtained by mixing PBS buffer and dimethyl sulfoxide (DMSO) in a volume ratio of 1: (4-10); preferably, a solvent obtained by mixing in a volume ratio of 1: 8.

[0019] The amount of the solvent used in step (1) is calculated based on 400-500 mL of solvent per mmol of sodium hyaluronate; preferably, it is calculated based on 450 mL of solvent per mmol of sodium hyaluronate.

[0020] The pH of the reaction in step (1) is 4.5 to 7.2, preferably pH=5.

[0021] The stirring reaction conditions in steps (1) and (2) are: stirring at 800 rpm at room temperature for 1 to 4 hours; preferably: stirring at 800 rpm at room temperature for 2 hours.

[0022] The conditions for the continued stirring reaction in steps (1) and (2) are: stirring at 800 rpm at room temperature for 12 to 24 hours; preferably: stirring at 800 rpm at room temperature for 24 hours.

[0023] The dialysis in step (1) is performed using a dialysis bag with a molecular weight cutoff of 3 kDa; preferably, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3 kDa for at least 72 hours, during which fresh dialysis fluid is continuously replaced to remove DMSO and excess 2,2'-dithiodiethylamine dihydrochloride, EDCI, and NHS.

[0024] The dialysate used for the dialysis in step (1) is water, preferably ultrapure water.

[0025] The freeze drying in steps (1) and (2) is performed at -40°C for 24 to 48 hours; preferably, the freeze drying is performed at -40°C for 36 hours.

[0026] The molar ratio of hemin, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and HA-SS-NH2 described in step (2) is 6:24-60:24-60:1; preferably 6:24:24:1 (Note: Since each Hemin contains two carboxyl groups and HA-SS-NH2 has 12 amino groups (one carboxyl group is grafted onto one amino group), the molar ratio of the carboxyl group in hemin, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the amino group in HA-SS-NH2 (i.e., the ratio of chemical reaction equivalents) is 1:2-5:2-5, 0.5-3, preferably 1:2:2:1).

[0027] The solvent described in step (2) is dimethyl sulfoxide (DMSO).

[0028] The amount of the solvent in step (2) is calculated based on 55-65 mL of solvent per mmol of hemin; preferably, it is calculated based on 62-63 mL of solvent per mmol of hemin.

[0029] The uniform mixing in step (2) is performed by ultrasonic mixing.

[0030] The water described in step (2) is ultrapure water or purified water.

[0031] The dropwise addition in step (2) is preferably performed by dropwise addition through a syringe, and the volume of the syringe can be selected according to actual needs. Preferably, a 1 mL syringe is used for dropwise addition.

[0032] The dialysis described in step (2) is a gradient dialysis, that is, dialysis is performed in dimethyl sulfoxide, a dialysate obtained by mixing dimethyl sulfoxide and water in a volume ratio of 1:1, and ultrapure water using a dialysis bag with a molecular weight cutoff of 5 kDa for at least 48 hours each.

[0033] The concentration in steps (2) and (3) is carried out by rotary evaporation.

[0034] The concentration of the HA-SS-Hemin polymer solution in step (3) is 10-20 mg / mL, preferably 10 mg / mL.

[0035] The amount of water (reverse phase solvent) used in step (3) will affect the particle size of the final product. Therefore, the amount of water used can be determined according to the critical micelle concentration (CMC); preferably, when the amount of HA-SS-Hemin polymer is 10 mg and the amount of DMSO is 1 mL, the amount of reverse phase solvent water used is in the range of 10 to 50 mL (preferably 10 mL).

[0036] The continuous stirring condition in step (3) is: stirring at 800 rpm at room temperature for 20 minutes to 1 hour; preferably: stirring at 800 rpm at room temperature for 1 hour.

[0037] The dialysis in step (3) is performed using a dialysis bag with a molecular weight cutoff of 10 kDa; preferably, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 10 kDa for at least 48 hours.

[0038] The dialysate used for the dialysis in step (3) is water; preferably ultrapure water.

[0039] The filtration in step (3) is performed using a 0.22 μm filter.

[0040] A hemin nano-micelle is prepared by any of the methods described above.

[0041] The hemin nano-micelles are spherical and have an average particle size of 60 nm.

[0042] The hemin nano-micelles are used in the preparation of a tumor cell pyroptosis inducer (i.e. a product that induces tumor cell pyroptosis).

[0043] The tumor includes breast cancer and colon cancer, etc.; preferably, breast cancer.

[0044] The use of the hemin nano-micelles (as a cell pyroptosis inducer) in the preparation of products for tumor immunotherapy.

[0045] The products include drugs, test kits, etc.

[0046] The tumor includes breast cancer and colon cancer, etc.; preferably, breast cancer.

[0047] The hemin chloride nano-micelles can inhibit the growth, proliferation and migration of tumor cells, induce pyroptosis of tumor cells, increase the total ROS level in cells, increase the Mito sox level in cells, reduce the expression of tumor cell mitochondrial membrane protein Tom20, reduce the mitochondrial membrane potential of tumor cells, promote tumor cells to release lactate dehydrogenase (LDH), induce tumor cells to secrete the inflammatory factor IL-1β, increase the number of M1 BMDMs and reduce the number of M2 BMDMs (with the ability to repolarize tumor-associated macrophages), increase the secretion of BMDM tumor suppressor factor TNF-α, promote DC maturation, induce its migration to tumor-draining lymph nodes, and increase the secretion of IFN-γ and TNF-α.

[0048] The application of the hemin nano-micelles (as a cell pyroptosis inducer) in the preparation of products for inhibiting the growth, proliferation and / or migration of tumor cells.

[0049] The tumor cells include breast cancer cells and colon cancer cells, etc.; preferably breast cancer cells.

[0050] The use concentration of the hemin nano-micelles is 12.5-100 μg / mL, preferably 50 μg / mL.

[0051] The products include drugs, test kits, etc.

[0052] Compared with the prior art, the present invention has the following advantages and effects:

[0053] (1) The present invention uses hemin chloride for the first time to develop a tumor-targeted pyroptosis inducer, using hemin chloride and hyaluronic acid as raw materials, which has the advantages of high biosafety, low cost and easy availability; the designed Hemin nanosystem is simple and the synthesis method is simple. The constructed new nano pyroptosis inducer has good biosafety, tumor targeting and responsiveness.

[0054] (2) The present invention utilizes hemin chloride nanoparticles to destroy the redox level in tumor cells, causing mitochondrial damage, thereby inducing pyroptosis of tumor cells, activating immune function, reshaping the immune microenvironment, and achieving tumor immunotherapy. At the same time, the present invention reveals the preliminary mechanism of hemin chloride nanoparticles inducing tumor cell pyroptosis, and further verifies its feasibility for tumor immunotherapy in an in situ 4T1 triple-negative breast cancer mouse model, providing a reference for the application of new pyroptosis inducers.

[0055] (3) The Hemin nanomicelles prepared in the present invention are uniform in size and have good stability. They can effectively kill tumor cells, effectively consume glutathione in tumor cells, and produce a large amount of reactive oxygen species, thereby destroying the intracellular redox balance and causing mitochondrial damage. At the same time, they can induce pyroptosis by activating the Caspase-3 / GSDME pathway, triggering immunogenic death effects, promoting the maturation and migration of dendritic cells, and repolarizing tumor macrophages, thereby producing a good tumor immunotherapy effect, providing a new strategy for the use of nanomedicines in pyroptosis / tumor immunotherapy synergistic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the ultraviolet absorption spectrum of the HA-SS-hemin polymer obtained in Example 1.

[0057] Figure 2 This is a diagram of the hydrated particle size of the Hemin nanomicelles obtained in Example 1.

[0058] Figure 3 This is the surface charge diagram of the Hemin nanomicelles obtained in Example 1.

[0059] Figure 4 This is a transmission electron microscope photograph of the Hemin nanomicelles obtained in Example 1.

[0060] Figure 5 This is a graph showing the particle size stability of the Hemin nanomicelles obtained in Example 1.

[0061] Figure 6 This is a graph showing the killing effect of Hemin nano-micelles of different concentrations on tumor cells 4T1 and MC38.

[0062] Figure 7This is a graph showing the killing effects of Hemin nano-micelles at different concentrations on normal cells 3T3-NIH and HUVEC.

[0063] Figure 8 This is a diagram showing the effect of Hemin nanomicelles on 4T1 cell clone formation.

[0064] Fig. 9 This is a diagram showing the effect of Hemin nanomicelles on the migration ability of 4T1 cells.

[0065] Fig.10 This is the flow cytometry result of the effect of Hemin nano-micelles on 4T1 cell apoptosis.

[0066] Fig.11 This is a statistical result diagram of the effect of Hemin nanomicelles on the glutathione level in 4T1 cells.

[0067] Fig.12 This is the killing result of Hemin nano-micelles combined with GSH in 4T1 cells.

[0068] Fig.13 This is a fluorescence image showing the effect of Hemin nanomicelles on the level of ROS in 4T1 cells.

[0069] Fig.14 This is the flow cytometry result of the effect of Hemin nanomicelles on the ROS level in 4T1 cells.

[0070] Fig.15 This is the killing result of Hemin nano-micelles in 4T1 cells after combined with NAC.

[0071] Fig.16 This is a fluorescence image showing the effect of Hemin nanomicelles on the Mito sox level in 4T1 cells.

[0072] Fig.17 This is the flow cytometry detection result of the effect of Hemin nanomicelles on the Mito sox level in 4T1 cells.

[0073] Fig.18 This is the western blot detection image of Tom20 protein in 4T1 cells after Hemin nanomicelle treatment.

[0074] Fig.19 This is the flow cytometry result of the effect of Hemin nanomicelles on mitochondrial membrane potential in 4T1 cells.

[0075] Fig. 20 This is the result of microscopic observation of 4T1 cell morphology after treatment with Hemin nano-micelles of different concentrations.

[0076] Fig.21 This is a statistical graph showing the effect of different concentrations of Hemin nanomicelles on the release of LDH from 4T1 cells.

[0077] Fig. 22 This is the killing result of Hemin nano-micelles combined with Z-VAD-FMK in 4T1 cells.

[0078] Fig.23 This is the western blot detection image of GSDME, GSDME-N, and Cleaved-caspase-3 proteins in 4T1 cells after Hemin nanomicelle treatment.

[0079] Fig.24 This is the ELISA test result of IL-1β secretion by 4T1 cells after being treated with different concentrations of Hemin nanomicelles.

[0080] Fig.25 This is a statistical graph of ATP release from 4T1 cells after treatment with different concentrations of Hemin nanomicelles.

[0081] Fig.26 It is the flow cytometry results of CRT expression in 4T1 cells after Hemin nano-micelle treatment and its average fluorescence intensity statistics.

[0082] Fig. 27 It is the flow cytometry results and statistical analysis diagram of the co-expression of CD80 and CD86 in BMDC cells after Hemin nano-micelle treatment.

[0083] Fig.28 It is the flow cytometry results and statistical analysis diagram of the co-expression of MHC-II and CD40 in BMDC cells after Hemin nano-micelle treatment.

[0084] Fig.29 This is a diagram showing the effect of Hemin nanomicelle treatment on BMDC cell migration ability.

[0085] Fig.30 This is the flow cytometry result of BMDM cell polarization after Hemin nanomicelle treatment.

[0086] Fig.31 This is a flow cytometric chart of BMDM cell polarization after treatment with Hemin nanomicelles.

[0087] Fig.32 This is the ELISA test result of TNF-α secretion in BMDM cells after Hemin nano-micelle treatment.

[0088] Fig.33This is the result of serum test after mice were injected with Hemin nano-micelles (in the figure, ALT: alanine aminotransferase; AST: aspartate aminotransferase).

[0089] Fig.34 This is a graph showing the results of serum testing of mice after injection of Hemin nano-micelles (in the graph, CREA: blood urea nitrogen; UREA: serum creatinine).

[0090] Fig.35 This is the result of H&E staining of mouse organs after injection of Hemin nano-micelles.

[0091] Fig.36 This is a statistical graph showing weight changes in 4T1 tumor-bearing mice treated with Hemin nano-micelles.

[0092] Fig.37 This is a graph showing the changes in tumor volume in 4T1 tumor-bearing mice treated with Hemin nano-micelles.

[0093] Fig.38 This is a photo of the tumor in 4T1 tumor-bearing mice after treatment with Hemin nano-micelles.

[0094] Fig.39 This is a graph showing the tumor weight of 4T1 tumor-bearing mice after treatment with Hemin nanomicelles.

[0095] Fig.40 This is a flow cytometry result diagram and its statistical result diagram of the expression of CD80 and CD86 of DC cells in the lymph node tissue of 4T1 tumor-bearing mice after Hemin nano-micelle treatment.

[0096] Fig.41 This is a flow cytometry result diagram and its statistical result diagram of the expression of MHC-II and CD40 of DC cells in the lymph node tissue of 4T1 tumor-bearing mice after Hemin nano-micelle treatment.

[0097] Fig.42 This is a statistical graph of the immune cells in the tumor tissue of 4T1 tumor-bearing mice after Hemin nano-micelles were used to treat the tumor.

[0098] Fig.43 This is the ELISA test result of IFN-γ in the serum of 4T1 tumor-bearing mice after Hemin nano-micelle treatment.

[0099] Fig.44 This is the ELISA test result of TNF-α in the serum of 4T1 tumor-bearing mice after Hemin nano-micelle treatment. DETAILED DESCRIPTION

[0100] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0101] Example 1. Preparation and characterization of Hemin nanomicelles

[0102] (1) Preparation of Hemin Nanomicelles

[0103] ① Synthesis of HA-SS-NH2: Sodium hyaluronate (MW = 4980, 200.0 mg, 0.04 mmol) was weighed and dissolved in 18 mL PBS / DMSO (1:8, v / v), pH was adjusted to 5, N-hydroxysuccinimide (166.4 mg, 1.446 mmol; NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (277.2 mg, 1.446 mmol; EDCI) were added, and after dissolution, stirring was continued at room temperature at 800 rpm for 2 hours; then 2,2'-dithiodiphenylamine was added. Ethylamine dihydrochloride (purchased from Bidex Pharmaceuticals, catalog number BD123147; CAS number: 56-17-7) (325.64 mg, 1.446 mmol) was dissolved in the above solution and stirred at 800 rpm at room temperature for 24 hours. After the reaction, it was dialyzed in pure water for 72 hours (room temperature) through a dialysis bag with a molecular weight cutoff of 3 kDa to remove DMSO and excess 2,2'-dithiodiethylamine dihydrochloride, EDCI, and NHS. The mixture was freeze-dried at -40°C for 36 hours to obtain HA-SS-NH2 white powder, which was stored at -20°C for use.

[0104] ② Synthesis of HA-SS-Hemin polymer: Weigh hemin (Hemin, MW = 651.94, 208.0 mg, 0.319 mmol) and dissolve it in 20 mL DMSO, add N-hydroxysuccinimide (147.4 mg, 1.28 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (245.4 mg, 1.28 mmol), and stir at room temperature for 2 hours after dissolution to obtain a Hemin solution; take 397.0 mg of the synthesized in step ① above HA-SS-NH2 powder was dissolved in 5 mL of ultrapure water. In an ice-water bath ultrasonic environment (68 kHz), the HA-SS-NH2 solution was slowly added dropwise to the Hemin solution through a 1 mL syringe and mixed evenly. Stirring was continued at 800 rpm at room temperature for 24 hours. After the reaction, the mixture was dialyzed in DMSO, DMSO:H2O (1:1, v / v), and ultrapure water for 48 hours using a dialysis bag with a molecular weight cutoff of 5 kDa. The mixture was collected, concentrated by evaporation on a rotary evaporator, freeze-dried at -40°C for 36 hours, and stored at -20°C for use.

[0105] ③Synthesis of Hemin nanomicelles: Nanoparticles were synthesized by nanoprecipitation method, i.e., 10 mg of HA-SS-Hemin polymer synthesized in step ② was dissolved in 1 mL of DMSO, and the mixture was dropped into 10 mL of ultrapure water with continuous stirring (800 rpm) through a 1 mL syringe, and stirred at 800 rpm at room temperature for 1 hour. The solution containing the nanoparticles was then transferred to a dialysis bag with a molecular weight cutoff of 10 kDa, and dialyzed in ultrapure water for 48 hours to remove DMSO. The dialyzed nanomicelle dispersion was concentrated by rotary evaporation, and the aqueous solution was finally filtered with a 0.22 μm filter to obtain Hemin nanomicelles, which were stored at 4°C for later use.

[0106] (2) Characterization of Hemin Nanomicelles

[0107] ① UV-visible spectroscopy: The spectral properties of HA-SS-Hemin polymer were analyzed by UV-visible spectrophotometer. The specific operation is as follows: Weigh the HA-SS-Hemin polymer and dissolve it in DMSO, dilute it into a series of test sample solutions with concentrations such as 100μg / mL, 50μg / mL, 25μg / mL, and 12.5μg / mL. Similarly, Hemin was prepared into test standard solutions with different concentration gradients (as a control) through the same operation as above. Use a UV-visible spectrophotometer to detect the characteristic peaks of the test solution, and analyze its spectral properties by comparing the characteristic absorption peaks of the standard to verify the successful synthesis of the polymer. The experiment was repeated three times. The characterization results of HA-SS-Hemin polymer are shown in the figure. Figure 1As shown, Hemin has a characteristic absorption peak at 392nm, and the UV absorption spectrum of the modified HA-SS-Hemin polymer can see a similar characteristic peak at 392nm. The UV-vis results indicate the successful modification of Hemin in the HA-SS-Hemin polymer.

[0108] ② Particle size and potential detection: a) Hemin nanomicelles were dispersed in PBS phosphate buffer (pH = 7.4) at a concentration of 1 mg / mL, added to the particle size measurement cell, and the particle size was detected by a nanoparticle size analyzer. Figure 2 As shown in the figure, the particle size of the obtained Hemin nano-micelles is mainly distributed in 60nm, and the dispersion is good. b) The Hemin nano-micelles are dispersed in PBS phosphate buffer (pH=7.4) at a concentration of 0.1mg / mL, added to a potential measurement cell, and the Zeta potential is measured using a nano-particle size analyzer. The results are shown in the figure. Figure 3 As shown, the micelle surface has a negative charge, the zata potential is around -18.9 mV, and there is an obvious Tyndall effect under light, indicating the successful synthesis of the nanoparticles.

[0109] ③ Morphological characterization: Hemin nano-micelles were dispersed in PBS buffer at a concentration of 0.1 mg / mL, 10 μL of the solution was added dropwise to a copper mesh coated with a carbon film, and after natural drying, the morphology of the nano-micelles was observed and photographed using a transmission electron microscope at 200 kV. The transmission electron microscope results are shown in Figure 2. Figure 4 As shown, Hemin nanomicelles present a uniform spherical morphology and the size is maintained at about 60 nm.

[0110] ④ Stability analysis: Hemin nano-micelles were dispersed in a simulated physiological environment (PBS phosphate buffer (pH = 7.4) containing 10% (v / v) fetal bovine serum (FBS)) at a concentration of 1 mg / mL, placed in an electric constant temperature oscillation tank at 37°C for 7 days, and samples were taken every 24 hours and added to the particle size measurement pool. The particle size change of the micelles was detected by a nano-particle size analyzer to analyze the stability of the nano-micelles. The experiment was repeated three times. The results are shown in Figure 5 As shown, the particle size of Hemin nanomicelles remained basically stable within seven days without significant changes, indicating that it has good stability over a period of time and is conducive to long-term storage.

[0111] Example 2: Detection of the toxicity of Hemin nanomicelles in different cell lines

[0112] 4T1 cells, MC38 cells, 3T3-NIH cells, and HUVEC cells (all purchased from ATCC) were seeded in 96-well cell culture plates at a density of about 1 to 2 × 10 4cell / well, culture overnight, and set aside. Before adding samples, the cells were first replaced with fresh DMEM high-glucose medium (purchased from Hyclone), and then Hemin nano-micelles (prepared in Example 1) were added at final concentrations of 12.5μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, and 200μg / mL, respectively. The negative control was an equal volume of PBS buffer. After 24 hours of cell culture, MTT cell viability detection was performed. The experiment was set up for three replicates.

[0113] The results are as follows Figure 6 and Figure 7 As shown, Hemin nano-micelles have obvious killing effects on 4T1 triple-negative breast cancer and MC38 colon cancer tumor cells, but at the same concentration, the toxicity to normal cells such as 3T3-NIH fibroblasts and HUVEC umbilical vein endothelial cells is significantly weakened, indicating that Hemin nano-micelles have good biosafety.

[0114] Example 3: Detection of the effect of Hemin nano-micelles on the proliferation and migration ability of 4T1 cells

[0115] (1) Cell clone formation experiment: 4T1 cells were counted, and a suspension of about 1000 single cells was inoculated into a 6-well cell culture plate and shaken evenly. After 24 hours of cell attachment, Hemin nanomicelles (NPs) with a final concentration of 50 μg / mL were added for 24 hours. The negative control was an equal volume of PBS buffer. The supernatant was then aspirated, and the cells were gently rinsed three times by adding 1× PBS buffer along the wall. After washing the drug solution, fresh culture medium was replaced and culture was continued for one week. During this period, observation was maintained and the culture medium was replaced. When the number of cell clones in the control wells was large, 1 mL of 4% paraformaldehyde fixative was added to each well after rinsing with PBS, and the cells were fixed at room temperature for 15 minutes. Subsequently, 1× PBS buffer was added along the wall to gently wash twice, and 2 mL of 0.1% crystal violet staining solution was added. The cells were stained at room temperature overnight, and the staining solution was recovered and rinsed with clean water several times until there was no obvious blue in the rinse solution. After being inverted to dry, the culture plate was placed on a white background to capture and save the image. The experiment was repeated three times. The results are as follows Figure 8 As shown, Hemin nano-micelles can effectively inhibit the proliferation and growth of cancer cells after treatment.

[0116] (2) Cell scratch test: 4T1 cells were inoculated into a 24-well cell culture plate at a rate of 3 to 5 × 10 4cell / well, culture overnight. The next day, when the cell monolayer density reached about 80%, draw a straight line in the center of the cell layer with a 200μL pipette tip, discard the supernatant and rinse three times with 1×PBS buffer, add fresh culture medium, and take pictures under a microscope to obtain the scratch situation at 0 hours. Then add Hemin nanomicelles with a final concentration of 50μg / mL for treatment for 24 hours. The negative control is to add an equal volume of PBS buffer. After the treatment, rinse three times with 1×PBS buffer, add fresh DMEM complete culture medium and continue to culture for 24 hours. Then, take pictures under a microscope to obtain the scratch situation at 48 hours, and analyze the migration ability of cancer cells in the treated group and the untreated group. The experiment was repeated three times. The results are as follows Fig. 9 As shown, Hemin nanomicelles can effectively inhibit the migration ability of cancer cells after treatment.

[0117] Example 4: Detection of the effect of Hemin nano-micelles on the apoptosis level of 4T1 cells

[0118] 4T1 cells were seeded in a 24-well cell culture plate at a density of about 3-5×10 4 cell / well, culture overnight and set aside. Before adding samples, cells were replaced with fresh DMEM high-glucose medium, and then treated with Hemin nano-micelles at a final concentration of 50 μg / mL. The negative control was an equal volume of PBS buffer. After 24 hours of cell culture, Annexin-V / PI flow cytometry was performed. The experiment was repeated three times. The results are shown in Fig.10 As shown, the late apoptosis level in the Hemin nano-micelle group increased from 5.8% to 59.6%, and the early apoptosis level also showed the same upward trend, indicating that Hemin nano-micelles can effectively induce tumor cell apoptosis.

[0119] Example 5: Detection of the effect of Hemin nano-micelles on intracellular glutathione levels in 4T1 cells

[0120] 4T1 cells were seeded in 6-well cell culture plates at a density of approximately 1×10 5 cell / well, culture overnight and set aside. Before adding samples, the cells were first replaced with fresh DMEM high-glucose medium, and then Hemin nano-micelles (prepared in Example 1) were added at final concentrations of 12.5μg / mL, 25μg / mL, 50μg / mL, and 100μg / mL, respectively. The negative control was an equal volume of PBS buffer. After 24 hours of cell culture, the intracellular glutathione level detection kit was used for detection. The experiment was repeated three times. The results are shown in Fig.11As shown, with the increase of Hemin nano-micelle treatment concentration, the GSH level in 4T1 cells gradually decreased. After 24 hours of treatment with 100 μg / mL Hemin nano-micelles, the GSH level decreased by 35%, indicating that the micelles have the ability to consume intracellular GSH.

[0121] 4T1 cells were seeded in a 96-well cell culture plate at a density of about 1-2×10 4 cell / well, culture overnight for later use. Before adding samples, replace the cells with fresh DMEM high-glucose medium, and then add Hemin nano-micelles with a final concentration of 30μg / mL, glutathione GSH with a final concentration of 2mM, Hemin nano-micelles with a final concentration of 30μg / mL and GSH with a final concentration of 2mM, respectively. The negative control (Control) is to add an equal volume of PBS buffer. MTT cell viability test was performed after 24 hours of treatment. The experiment was repeated three times. The results are shown in Fig.12 As shown, exogenously added GSH can reverse the killing effect of Hemin nanomicelles on 4T1 cells.

[0122] Example 6: Detection of the effect of Hemin nanomicelles on ROS levels in 4T1 cells

[0123] 4T1 cells were seeded in a 12-well cell culture plate at a density of approximately 5 × 10 4 cell / well, culture overnight for standby use. Before adding samples, the cells were first replaced with fresh DMEM culture medium, followed by the addition of Hemin nanomicelles (prepared in Example 1) with a final concentration of 50 μg / mL, Hemin solution with a final concentration of 50 μg / mL, Hemin nanomicelles with a final concentration of 50 μg / mL, and NAC (antioxidant, N-acetylcysteine, Sigma) with a final concentration of 5 mM. The blank control was an equal volume of PBS buffer. After 4 hours of treatment, the cells were washed twice with PBS buffer. Then fresh culture medium containing 10 μM DCFH-DA (2',7'-dichlorofluorescein diacetate) was added, incubated in a 37°C incubator away from light for 20 minutes, rinsed twice with PBS, replaced with fresh culture medium, and the fluorescence image at 488 nm was detected by fluorescence microscopy. After the same treatment was completed, trypsin digestion was performed, and FACS (flow cytometry) detection was performed after washing with PBS buffer. The experiment was repeated three times. The results are as follows Fig.13 and Fig.14 As shown, both the flow cytometry results and the fluorescence images showed that Hemin nanomicelles could increase the total ROS level in cells, and the increase of ROS could be inhibited by NAC.

[0124] 4T1 cells were seeded in a 96-well cell culture plate at a density of about 1-2×10 4cell / well, culture overnight for standby use. Before adding samples, the cells were first replaced with fresh DMEM high-glucose culture medium, followed by the addition of Hemin nanomicelles (prepared in Example 1) with a final concentration of 50 μg / mL, Hemin solution with a final concentration of 50 μg / mL, Hemin nanomicelles with a final concentration of 50 μg / mL, and NAC (antioxidant, N-acetylcysteine, Sigma) with a final concentration of 5 mM, respectively. The negative control was the addition of an equal volume of PBS buffer. MTT cell viability assay was performed after 24 hours of treatment. The experiment was repeated three times. The results are shown in Fig.15 As shown, the reactive oxygen species inhibitor NAC can effectively inhibit the killing effect of Hemin nanomicelles on 4T1 cells.

[0125] Example 7: Detection of the effect of Hemin nano-micelles on Mitosox levels in 4T1 cells

[0126] 4T1 cells were seeded in a 12-well cell culture plate at a density of approximately 5 × 10 4 cell / well, culture overnight for standby use. Before adding samples, replace the cells with fresh DMEM culture medium, and then add Hemin nanomicelles (prepared in Example 1) with a final concentration of 50μg / mL, a solution with a final concentration of 50μg / mL Hemin, nanomicelles with a final concentration of 50μg / mL Hemin, and NAC (antioxidant, N-acetylcysteine, Sigma) with a final concentration of 5mM, respectively. The blank control is an equal volume of PBS buffer. After treatment for 8 hours, wash the cells twice with 1×PBS buffer. Add Mito sox red fluorescent probe with a final concentration of 5μM and incubate at 37°C in the dark for 15 minutes. After the end, rinse twice with 1×PBS buffer and take pictures under a fluorescence microscope. In addition, after the same treatment, collect the cells and resuspend and filter them into a flow tube, and detect the intracellular Mito sox level by flow cytometry. The experiment was repeated three times. The results are as follows Fig.16 and Fig.17 As shown, both the flow cytometry results and the fluorescence imaging results showed that Hemin nanomicelles could increase the level of Mito sox in cells.

[0127] Example 8: Detection of the effect of Hemin nano-micelles on 4T1 cell mitochondria

[0128] (1) Detection of mitochondrial membrane protein Tom20: 4T1 cells were inoculated in a 24-well cell culture plate at a density of approximately 3×10 4cell / well, culture overnight and set aside. Before adding samples, replace the cells with fresh DMEM medium, then add Hemin nano-micelles with a final concentration of 25μg / mL, 50μg / mL, 100μg / mL and Hemin solution with a final concentration of 50μg / mL, respectively. The blank control is an equal volume of PBS buffer. Western blot detection was performed after 24 hours of treatment. The experiment was repeated three times. The results are shown in Fig.18 As shown, Hemin nanomicelles can downregulate the expression of mitochondrial membrane protein Tom20 in 4T1 cells.

[0129] (2) Mitochondrial membrane potential detection: 4T1 cells were inoculated in a 12-well cell culture plate at a density of approximately 5×10 4 cell / well, culture overnight for later use. Before adding samples, replace the cells with fresh DMEM culture medium, add Hemin nanomicelles and 50μg / mL Hemin solution with a final concentration of 50μg / mL respectively, and the blank control is an equal volume of PBS buffer. After 12 hours of treatment, the intracellular mitochondrial membrane potential was detected by the mitochondrial membrane potential detection kit (JC-1). Specifically, the supernatant was aspirated, rinsed twice with 1×PBS buffer, and 1×JC-1 staining working solution prepared with JC-1 staining buffer was added and incubated at 37°C in the dark for 20 minutes. After the end, rinsed twice with 1×PBS buffer, collected the cell pellet and resuspended it for flow cytometry detection. The experiment was repeated three times. The results are as follows Fig.19 As shown, Hemin nanomicelles can reduce the mitochondrial membrane potential of 4T1 cells.

[0130] Example 9: Detection of the effect of Hemin nano-micelles on 4T1 cell morphology

[0131] 4T1 cells were inoculated in a 35 mm cell glass dish at a density of about 3 to 5 × 10 4 cell / well, culture overnight and set aside. Before adding samples, cells were replaced with fresh DMEM high-glucose medium, and then treated with Hemin nano-micelles at final concentrations of 25μg / mL, 50μg / mL, and 100μg / mL, respectively. The negative control was an equal volume of PBS buffer. After 24 hours of cell culture, cell morphology changes were observed. The experiment was repeated three times. The results are shown in Fig. 20 As shown, after being treated with Hemin nano-micelles, 4T1 cells showed obvious vacuolar swelling and enlargement, and with the increase of micelle treatment concentration, the vacuolar characteristics of the cells became more obvious.

[0132] Example 10: Detection of LDH release from 4T1 cells after treatment with Hemin nanomicelles

[0133] 4T1 cells were seeded in a 96-well cell culture plate at a density of about 1-2×10 4 cell / well, culture overnight for later use. Remove the culture medium, rinse once with 1×PBS buffer, and then add Hemin nanomicelles with final concentrations of 12.5μg / mL, 25μg / mL, 50μg / mL, and 100μg / mL for treatment for 24 hours, with five replicate wells each. At the same time, set up background blank control wells, sample control wells, and sample maximum enzyme activity control wells. Subsequently, the release level of cell LDH was detected using a lactate dehydrogenase (LDH) cytotoxicity detection kit. The experiment was repeated three times. The results are as follows Fig.21 As shown, Hemin nanomicelles promoted the release of LDH from 4T1 cells. Under the intervention of 50 μg / mL Hemin nanomicelles, the release of LDH in the cell supernatant increased by about 7 times, which was concentration-dependent.

[0134] Example 11: Detection of cell death types caused by Hemin nanomicelles

[0135] 4T1 cells were seeded in a 96-well cell culture plate at a density of about 1-2×10 4 cell / well, culture overnight for later use. Before adding samples, replace the cells with fresh DMEM high-glucose medium, and then add Hemin nano-micelles with a final concentration of 25μg / mL, 25μg / mL Hemin nano-micelles + 50μM Z-VAD-FMK (Caspase inhibitor, MCE), and the negative control is to add an equal volume of 1×PBS buffer. MTT cell viability test was performed after 24 hours of treatment. The experiment was repeated three times. The results are shown in Fig. 22 As shown in the figure, after combined with the pyroptosis inhibitor Z-VAD-FMK, the killing effect of Hemin nanoemulsifier on 4T1 cells was significantly inhibited, which indirectly indicates that 4T1 cells underwent pyroptosis after treatment with Hemin nanoemulsifier.

[0136] Example 12: Detection of the cell pyroptosis pathway induced by Hemin nanomicelles

[0137] 4T1 cells were seeded in a 24-well cell culture plate at a density of approximately 3 × 10 4 cell / well, culture overnight and set aside. Before adding samples, cells were replaced with fresh DMEM medium, followed by addition of Hemin nano-micelles and 50μg / mL Hemin solution at a final concentration of 50μg / mL, respectively. The blank control was an equal volume of PBS buffer. After 24 hours of treatment, western blot was performed to detect the expression of pyroptosis-related marker proteins. The experiment was repeated three times. The results are shown in Fig.23As shown, after 24 hours of induction with Hemin nano-micelles at a final concentration of 50 μg / mL, the expression of Caspase-3 spliceosome in 4T1 cells was significantly upregulated compared with the untreated group, indicating that Caspase-3 was activated. At the same time, the expression of GSDME-N protein, the downstream pyroptosis execution molecule of Caspase-3, was significantly upregulated, and the expression of full-length GSDME protein was reduced, indicating that Hemin nano-micelles induce pyroptosis in 4T1 tumor cells by activating the Caspase-3 / GSDME pathway.

[0138] Example 13: Detection of 1L-1β release in 4T1 cells after treatment with Hemin nanomicelles

[0139] 4T1 cells were seeded in a 24-well cell culture plate at a density of approximately 3 × 10 4 cell / well, culture overnight, and set aside. Before adding samples, replace the cells with fresh DMEM medium, and then add Hemin nano-micelles with final concentrations of 12.5μg / mL, 25μg / mL, 50μg / mL, and 100μg / mL, respectively. The blank control is an equal volume of PBS buffer. After 24 hours of treatment, the cell culture fluid was collected and ELISA detection of IL-1β was performed after high-speed centrifugation at 4°C. The experiment was repeated three times. The results are shown in Fig.24 As shown, Hemin nanomicelles can induce 4T1 cells to secrete the inflammatory factor IL-1β.

[0140] Example 14: Detection of ATP secretion level of 4T1 cells after treatment with Hemin nanomicelles

[0141] 4T1 cells were seeded in 6-well cell culture plates at a density of approximately 1×10 5 cell / well, culture overnight for later use. When the density reaches 40-50%, before adding samples, replace the cells with fresh DMEM culture medium, then add Hemin nanomicelles with final concentrations of 25μg / mL, 50μg / mL, and 100μg / mL for treatment for 24 hours. The blank control is an equal volume of PBS buffer. Aspirate the culture medium and add 200μL lysis buffer to each well to lyse the cells. Centrifuge at 4℃ and 12000g for 5 minutes, and take the supernatant for measurement. The secretion of cellular ATP was detected by an ATP detection kit. The experiment was repeated three times. The results are as follows Fig.25 As shown, Hemin nano-micelles can promote 4T1 cells to secrete ATP to the extracellular space, and with the increase of drug concentration, the content of ATP in the supernatant of 4T1 tumor cell culture fluid also increases in a concentration-dependent manner.

[0142] Example 15: Detection of the effect of Hemin nano-micelles on the CRT expression level in 4T1 cells

[0143] 4T1 cells were seeded in a 12-well cell culture plate at a density of approximately 5 × 10 4 cell / well, culture overnight for standby use. Before adding samples, replace the cells with fresh DMEM medium. After 24 hours of cell attachment, add Hemin nano-micelles and 50μg / mL Hemin solution at a final concentration of 50μg / mL for 24 hours, respectively. The blank control is an equal volume of PBS buffer. Collect the cell supernatant and cell pellet, centrifuge at 1000g for 5 minutes at room temperature, prepare Anti-Calreticulin-antibody flow cytometry antibody (Anti-Calreticulin-antibody purchased from Cell Signaling Technology, catalog number 12238) with PBS containing 1% (v / v) FBS, resuspend each sample in 30μL volume, incubate on ice in the dark for 30 minutes, add 1mL of PBS containing 1% (v / v) FBS to rinse once, centrifuge at 1000g for 5 minutes at room temperature, resuspend the cells in 200μL PBS, and detect the expression of cell surface calreticulin (CRT) by flow cytometry. The experiment was repeated three times. The results are as follows Fig.26 As shown in the figure, Hemin nanomicelles significantly promoted the expression of CRT on the surface of 4T1 cells, and the same concentration of free Hemin could also regulate the exposure level of CRT to a certain extent.

[0144] Example 16: Detection of the ability of Hemin nanomicelles to activate dendritic cells

[0145] (1) Extraction and isolation of BMDC: 8-week-old C57BL / 6 mice (weight about 20 g) (male, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) were killed by dislocation. The femur and tibia of the mice were exposed under sterile operation, and the muscle tissue was removed. The bone marrow cavity was flushed with PBS buffer containing an appropriate amount of FBS, centrifuged and the culture medium was discarded. After lysing red blood cells, RPMI 1640 complete culture medium containing 10 ng / mL IL-4 and 25 ng / mL granulocyte macrophage colony stimulating factor GM-CSF was added and resuspended for culture. The mice were observed every day, and half of the culture medium containing sufficient nutrient factors was replaced on the third day. BMDCs can be collected on the fifth day and re-plated for subsequent experiments.

[0146] (2) BMDC maturity detection: 4T1 cells were inoculated in a 12-well cell culture plate at a density of approximately 5×10 4cell / well, culture overnight and set aside. When the cell confluence reached 50%, before adding samples, the cells were first replaced with fresh DMEM medium, and then Hemin nanomicelles and 25 μg / mL Hemin solution were added at a final concentration of 25 μg / mL for 24 hours, respectively. The blank control was an equal volume of PBS buffer. The cells were collected and counted to obtain drug-treated 4T1 cells. At the same time, the immature BMDCs in the above step (1) were collected and counted, and then the drug-treated 4T1 cells and BMDCs were co-incubated in a 24-well plate at a ratio of 1:2. After 24 hours of co-culture, the semi-suspended BMDCs were collected and blocked with blocking antibodies (Purified anti-mouse CD16 / 32 purchased from BioLegend) (1:100, v / v) at 4°C for 20 minutes. Flow cytometry antibodies were added for staining: CD45.2, CD11c, IA / IE, CD86, CD80, CD40 (APC / Cyanine7 anti-mouse CD45.2, PE anti-mouse CD11c, Alexa 700 anti-mouse IA / IE, Brilliant Violet 421 TM Anti-mouse CD86, APCanti-mouse CD80, FITC anti-mouse CD40, all purchased from BioLegend). The cells were stained at 4°C for 30 minutes, then rinsed with 1 mL of PBS, resuspended with 300 μL of PBS, and the maturity of BMDCs was detected by flow cytometry. The experiment was repeated three times. The results are shown in Fig. 27 and Fig.28 As shown, compared with the control group, the CD80 + CD86 + , the expression levels of antigen presenting molecule MHC-II and co-stimulatory molecule CD40 were significantly upregulated.

[0147] (3) BMDC migration ability: 4T1 cells were seeded in the lower layer of a Transwell 24-well plate at a density of approximately 3×10 4cell / well, culture overnight for standby use. When the cell fusion degree reaches 50%, then add Hemi nanomicelles with a final concentration of 50μg / mL and Hemin solution with a final concentration of 50μg / mL respectively. After treatment for 24 hours, collect BMDC and count them, add 200,000 BMDC cells to the Transwell chamber, incubate for 24 hours, transfer the chamber to a new well, add 500μL RPMI 1640 complete medium containing chemokine CCL21 (recombinant mouse CCL21 protein purchased from Abcam) (1:1000, v / v) to the well, incubate for 2 hours, then take out the chamber, gently wipe off the upper layer of cells with a cotton swab, fix with 4% paraformaldehyde for 15 minutes, stain with 0.1% crystal violet solution at room temperature for 2 hours, rinse with water several times and continue to gently wipe off the residual cells on the upper layer of the chamber, observe and photograph the migration of DC with an inverted microscope, and save the image. The experiment was repeated three times. The results are as follows Fig.29 As shown, compared with the control group, the number of BMDC cells in the Hemin nanomicelle group that migrated to the bottom of the chamber increased significantly, indicating that their migration ability was improved.

[0148] Example 17: Detection of the ability of Hemin nanomicelles to repolarize tumor-associated macrophages

[0149] (1) Extraction and separation of BMDM: 8-week-old C57BL / 6 mice (weight about 20 g) (male, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) were killed by dislocation. The femur and tibia of the mice were exposed under sterile operation, and the muscle tissue was removed. The bone marrow cavity was washed with PBS buffer containing an appropriate amount of FBS, centrifuged and the culture medium was discarded. After lysing the red blood cells, DMEM complete culture medium containing 10% (v / v) L929 cell supernatant was added and resuspended, and cultured in a 100 mm cell culture dish. The cells were observed every day. The adherent cells were BMDM and could be used for experiments on the fifth day.

[0150] (2) BMDM polarity detection: BMDM cells were seeded in a 6-well cell culture plate. After overnight culture, IL-4 with a final concentration of 10 ng / mL was added for stimulation for 12 hours. IFN-γ and LPS were added to the control well (M1) for stimulation for 12 hours. Then, the culture medium was replaced and Hemin nanomicelles with a final concentration of 10 μg / mL were added for treatment for 12 hours. After the treatment, the cells were digested with trypsin, collected, and blocked with blocking antibodies at 4°C for 20 minutes. Then, flow cytometry antibodies were added for staining: CD11b, F4 / 80, CD206, CD86 (PE anti-mouse CD11b, PE / Cyanine7 anti-mouse F4 / 80, APC anti-mouse CD206, BrilliantViolet 421 TMAnti-mouse CD86, both purchased from BioLegend). The cells were stained at 4°C for 30 minutes, and then rinsed with 1 mL of PBS. Finally, the cells were resuspended in 300 μL of PBS and the polarization state of macrophages was detected by flow cytometry. The experiment was repeated three times. The results are shown in Fig.30 and Fig.31 As shown, after treatment with Hemin nanomicelles, the number of M2 BMDMs decreased and the number of M1 BMDMs increased, indicating that Hemin nanomicelles have the ability to repolarize tumor-associated macrophages.

[0151] (3) Detection of tumor suppressor TNF-α: BMDM cells were seeded in a 24-well cell culture plate. After overnight culture, Hemin nano-micelles with a final concentration of 25 μg / mL and 100 ng / mL lipopolysaccharide (LPS, positive control) were added for 12 hours. The blank control was an equal volume of PBS buffer. After the end, the cell culture supernatant was collected and the secretion of TNF-α factor was detected by ELISA. The experiment was repeated three times. The results are shown in Fig.32 As shown in the figure, after treatment with Hemin nanomicelles, the secretion of tumor suppressor TNF-α in BMDM was significantly upregulated.

[0152] Example 18: Testing the biosafety of Hemin nanomicelles

[0153] (1) Liver and kidney function indicators: BALB / c mice (weight about 20g) (female, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) were divided into three groups, three mice in each group. The treatment group was injected with 20mg / kg Hemin nano-micelles (prepared in Example 1) and Hemin solution by tail vein, and the negative control was injected with an equal volume of PBS buffer. After 24 hours, blood was collected from the orbital venous plexus, and serum was separated to detect the concentrations of AST (aspartate aminotransferase), ALT (alanine aminotransferase), UREA (blood urea nitrogen), and CREA (serum creatinine) in the serum. The results are shown in Figure 2. Fig.33 and Fig.34 As shown, compared with the PBS group, there was no significant difference in the liver and kidney function indicators between the Hemin nano-micelles treatment group and the PBS group, indicating that Hemin nano-micelles would not cause significant effects on the internal organs of mice.

[0154] (2) HE staining of tissue sections: BALB / c mice (weighing about 20 g) were divided into three groups, with three mice in each group. The treatment group was injected with 20 mg / kg of Hemin nanomicelles (prepared in Example 1) and Hemin solution through the tail vein, and the negative control was injected with an equal volume of PBS buffer. The mice were sacrificed 24 hours later, and the heart, liver, spleen, lung, kidney and other important organs of the mice were taken for HE staining observation. The results are shown in Figure 2. Fig.35As shown, the texture of each tissue in the Hemin nano-micelle treatment group was not significantly different from that in the PBS control group, which also shows that Hemin nano-micelles will not cause obvious damage to the internal organs of mice.

[0155] Example 19: Detection of the in vivo anti-tumor immune effect of Hemin nanomicelles

[0156] (1) BALB / c mice (weight about 22 g) (female, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) were injected with 0.1 mL of 4T1 cell suspension (1 million) into the right mammary pad to establish an orthotopic 4T1 tumor model. When the tumor volume grew to 100 mm 3 At the same time, the tumor-bearing mice were randomly divided into three groups (n=6) to start the treatment experiment. The groups were: PBS control group, free Hemin group, and Hemin nano-micelle group. On days 0, 3, 6, and 9, PBS buffer, free Hemin solution, and Hemin NPs were injected into the mice through the tail vein, respectively. The dosage was 20 mg / kg, and a total of 4 doses were given. The treatment cycle was 12 days. The body weight and tumor volume of the tumor-bearing mice were measured and recorded every other day to observe the tumor growth. The results are as follows Fig.36 , Fig.37 , Fig.38 , Fig.39 As shown, compared with the control group, the weight of mice did not decrease significantly after treatment with Hemin nanoemulsifiers. From the tumor growth curve, tumor volume and size changes, it can be seen that Hemin nanoemulsifiers have obvious anti-tumor effects, and the effect is better than that of free Hemin, which reflects the advantages of this nanosystem.

[0157] (2) Lymph node immunoassay: After the treatment, sacrifice BALB / c tumor-bearing mice, remove the draining lymph node tissue, grind it, collect the cell suspension and centrifuge it. Discard the supernatant, add 1 mL of red blood cell lysis buffer (purchased from Biosharp), gently resuspend the cells, lyse at room temperature for 5 minutes, add 2 mL of PBS to neutralize after lysis, and centrifuge at 450g for 5 minutes. Discard the supernatant, add 1 mL of PBS to resuspend the cells and transfer them to a 1.5 mL centrifuge tube for cell counting. Take 1×10 6 The cells were blocked with blocking antibodies on ice for 15 minutes, and then CD45.2, CD11c, CD40, IA / IE, CD80 and CD86 (APC / Cyanine7 anti-mouse CD45.2, PE anti-mouse CD11c, Alexa 700anti-mouse IA / IE、Brilliant Violet 421 TMAnti-mouse CD86, APC anti-mouse CD80, and FITC anti-mouse CD40 were all purchased from BioLegend. Antibody dilutions were prepared at a certain dilution concentration for staining and incubated at 4°C in the dark for 30 minutes. Then 1 mL of PBS was added to rinse the cells and centrifuged at 3000 g for 3 minutes. Finally, 300 μL of PBS was added to resuspend the cells, filtered through a 200-mesh nylon filter into a flow tube, and detected on an 18-color flow cytometer. The results are shown in Fig.40 and Fig.41 As shown, after treatment with Hemin nano-micelles, mature DCs (CD80 + CD86 + ) was significantly increased, from an average of 9.56% in the PBS group to an average of 21.23% in the Hemin nano-micelle treatment group. Free Hemin treatment can also stimulate DC to a certain extent, with an average mature DC ratio of 13.35%. At the same time, Hemin nano-micelle treatment can significantly upregulate the expression of co-stimulatory molecules MHC II and CD40 on the surface of DC, and the proportion of simultaneous expression of MHC II and CD40 molecules increased from 24.88% in the PBS control group to 42.78% in the Hemin NPs group, which is statistically significant; free Hemin treatment can also increase the co-expression of MHC II and CD40 molecules on the surface of DC to a certain extent, but the effect of Hemin NPs is more obvious. The above data show that Hemin nano-micelles promote DC maturation in vivo and induce its migration to tumor-draining lymph nodes.

[0158] (3) Tumor tissue immune analysis: After the treatment, BALB / c tumor-bearing mice were sacrificed, the tumors were removed, 4 mL of collagenase IV (1 mg / mL, prepared in basal medium DMEM) was added, the tumor tissue was chopped up and placed in a 37°C shaker for digestion at 180 rpm for 1 hour. After digestion, the tumor tissue suspension was filtered using a 200-mesh nylon filter and transferred to a 15 mL centrifuge tube, centrifuged at 450 g for 5 minutes, and the supernatant was discarded. 7 mL of 40% Percoll solution was added to resuspend the cells, and centrifuged at 800 g for 20 minutes at room temperature to separate tumor cells and immune cells. The upper layer of liquid was then removed, and 2 mL of red blood cell lysis buffer was added to resuspend the cells, and the cells were lysed at room temperature for 7 minutes. After the end, PBS was filled to neutralize the red blood cell lysis buffer, centrifuged at 450 g for 5 minutes, the supernatant was discarded, 1 mL of PBS was added to resuspend the cells and transferred to a 1.5 mL centrifuge tube for cell counting. 1×10 6cells, add antibodies and block on ice for 15 minutes. After blocking, add CD45.2, CD11b, IA / IE, CD3, CD4, CD8, CD44, CD62L, CD206, CD86, F4 / 80 antibodies (APC / Cyanine7anti-mouse CD45.2, PE anti-mouse CD11b, Alexa 700anti-mouse IA / IE, PEanti-mouse CD3, Brilliant Violet 785 TM anti-mouse CD8, Brilliant Violet 510 TM anti-mouse CD44, FITC anti-mouse CD62L, APC anti-mouse CD206, Brilliant Violet421 TM Anti-mouse CD86, PE / Cyanine7 anti-mouse F4 / 80 were purchased from BioLegend; BUV563 anti-mouse CD4 was purchased from BD Biosciences) was used for staining with diluent and incubated at 4°C in the dark for 30 minutes. Then 1 mL of PBS was added to rinse the cells and centrifuged at 3000 g for 3 minutes. Finally, 300 μL of PBS was added to resuspend the cells, filtered through a 200-mesh nylon filter and transferred to a flow tube, and detected on an 18-color flow cytometer. The results are shown in Fig.42 As shown, compared with the PBS group, the CD4 + T cells and CD8 + T cell infiltration increased significantly, and CD4 + T cells and CD8 + Central memory T cells (T EM ) and effector memory T cells (T CM The ratio of M1 / M2 macrophages in tumor tissues was further analyzed, and the number of regulatory T cells (Treg) with immunosuppressive effects decreased, indicating that Hemin nano-micelles can not only activate CD8 +T cells can play a tumor-killing role and induce tumor-associated macrophages to repolarize from M2 to M1, reversing the immunosuppressive state and reshaping the immune microenvironment. (4) Detection of inflammatory factors in serum: After the treatment of mice, blood was collected from the orbital venous plexus, serum was separated, and the levels of IFN-γ and TNF-α in the serum were detected by ELISA kit. The average value of three measurements was taken. The results are shown in Fig.43 and Fig.44 As shown, compared with the control group, Hemin nano-micelles treatment can significantly up-regulate the secretion of IFN-γ and TNF-α in the body, indicating that Hemin nano-micelles have the function of activating immunity.

[0159] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing hemin nanomicelles, characterized in that: The steps include: (1) Synthesis of HA-SS-NH2: Dissolve sodium hyaluronate in a solvent, then add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir to react, then add 2,2'-dithiodiethylamine dihydrochloride, continue to stir to react, dialyze after the reaction is completed, and freeze-dry to obtain HA-SS-NH2 powder; (2) Synthesis of HA-SS-Hemin polymer: Dissolve hemin in a solvent, then add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and stir to react, to obtain a Hemin solution; dissolve the HA-SS-NH2 powder obtained in step (1) in water to obtain a HA-SS-NH2 solution; then slowly drop the HA-SS-NH2 solution into the Hemin solution, mix well, and continue to stir to react, after the reaction is completed, dialyze, concentrate, and freeze-dry to obtain a HA-SS-Hemin polymer; (3) Synthesis of Hemin Nanomicelles: The HA-SS-Hemin polymer obtained in step (2) is dissolved in dimethyl sulfoxide to obtain a HA-SS-Hemin polymer solution; then the HA-SS-Hemin polymer solution is added dropwise into continuously stirred water, stirred for reaction, dialyzed, concentrated, and filtered to obtain hemin nano-micelles.

2. The method for preparing hemin nano-micelles according to claim 1, characterized in that: The molar ratio of sodium hyaluronate, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2,2'-dithiodiethylamine dihydrochloride described in step (1) is 1:36-60:36-60:36-60; The molar ratio of hemin, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and HA-SS-NH2 in step (2) is 6:24-60:24-60:1; The concentration of the HA-SS-Hemin polymer solution described in step (3) is 10-20 mg / mL.

3. The method for preparing hemin nano-micelles according to claim 2, characterized in that: The molar ratio of sodium hyaluronate, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2,2'-dithiodiethylamine dihydrochloride described in step (1) is 1:36:36:36; The molar ratio of hemin, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and HA-SS-NH2 in step (2) is 6:24:24:1; The concentration of the HA-SS-Hemin polymer solution described in step (3) is 10 mg / mL.

4. The method for preparing hemin nano-micelles according to claim 1, characterized in that: The weight average molecular weight of the sodium hyaluronate described in step (1) is 4980; The solvent in step (1) is a solvent obtained by mixing PBS buffer and dimethyl sulfoxide in a volume ratio of 1:4-10; The pH of the reaction in step (1) is 4.5 to 7.2; The dialysis in step (1) is performed using a dialysis bag with a molecular weight cut-off of 3 kDa; The dialysate used in the dialysis described in steps (1) and (3) is water; The solvent described in step (2) is dimethyl sulfoxide; The dialysis in step (2) is a gradient dialysis: the dialysate is sequentially dialyzed in dimethyl sulfoxide, a dialysate obtained by mixing dimethyl sulfoxide and water in a volume ratio of 1:1, and ultrapure water using a dialysis bag with a molecular weight cutoff of 5 kDa for at least 48 hours each; The dialysis described in step (3) is performed using a dialysis bag with a molecular weight cut-off of 10 kDa.

5. The method for preparing hemin nano-micelles according to claim 1, characterized in that: The stirring reaction conditions in steps (1) and (2) are: stirring at 800 rpm at room temperature for 1 to 4 hours; The conditions for continuing the stirring reaction in steps (1) and (2) are: stirring at room temperature and 800 rpm for 12 to 24 hours; The freeze drying in steps (1) and (2) is freeze drying at -40°C for 24 to 48 hours; The concentration in steps (2) and (3) is carried out by rotary evaporation; The continuous stirring conditions in step (3) are: stirring at 800 rpm at room temperature for 20 minutes to 1 hour; The filtration in step (3) is performed using a 0.22 μm filter.

6. A hemin nano-micelle, characterized in that: It is prepared by the method according to any one of claims 1 to 5.

7. Use of the hemin nano-micelles according to claim 6 in the preparation of a tumor cell pyroptosis inducer.

8. Use of the hemin nano-micelles according to claim 6 in the preparation of products for tumor immunotherapy.

9. Use of the hemin nano-micelles according to claim 6 in the preparation of products for inhibiting the growth, proliferation and / or migration of tumor cells.

10. The use according to any one of claims 7 to 9, characterized in that: The tumor is at least one of breast cancer and colon cancer.

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