CN113995854A - Glutathione-sensitive nano drug delivery system and preparation method and application thereof - Google Patents
Glutathione-sensitive nano drug delivery system and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a glutathione sensitive nano drug delivery system and a preparation method and application thereof. The nano drug delivery system is obtained by taking a metal organic framework compound Cu-BTC as a carrier and encapsulating indoleamine 2, 3-dioxygenase inhibitor and nitric oxide donor. A nano metal organic framework compound Cu-BTC sensitive to glutathione is adopted as a nano carrier to mediate nitric oxide and micromolecular indoleamine-2, 3-dioxygenase inhibitor to carry out synergistic immunotherapy on tumors, the indoleamine-2, 3-dioxygenase inhibitor and nitric oxide synergistically regulate tumor immunosuppressive microenvironment, the content of cytotoxic T lymphocytes is obviously improved, and the number of regulatory T cells is reduced, so that the efficient tumor immunotherapy effect is realized, the problem of limited effect of immunotherapy by independently using the indoleamine-2, 3-dioxygenase inhibitor in the prior art is solved, and a novel and efficient therapeutic drug is provided for immunotherapy on solid tumors.
Description
Technical Field
The invention belongs to the technical field of antitumor drug delivery, and particularly relates to a glutathione-sensitive nano drug delivery system and a preparation method and application thereof.
Background
Immunotherapy has shown strong antitumor activity in the clinical treatment of various solid tumors such as melanoma, Renal Cell Carcinoma (RCC) and non-small cell lung cancer (NSCLC). The tumor immunotherapy induces an organism to generate tumor specific immune response in an active or passive mode, inhibits tumor growth and prevents tumor recurrence or metastasis. The effective tumor immunotherapy strategies reported to date mainly include Immune Checkpoint Blockade (ICB), adoptive T cell therapy (ACT), tumor-specific vaccines, and the use of small molecule immunomodulatory drugs.
Among the numerous therapeutic strategies, inhibition of indoleamine-2, 3-dioxygenase (IDO) is of widespread interest as it plays a critical role in mediating tumor immune escape. Indoleamine-2, 3-dioxygenase catalyzes the degradation of tryptophan (Trp) to kynurenine (Kyn), which inhibits the function of Cytotoxic T Lymphocytes (CTL) and activates regulatory T cells (tregs). The strong immunomodulatory function of indoleamine-2, 3-dioxygenase is attributed to tryptophan "starvation" and accumulation of kynurenine levels. Compared with the conventional treatment means, indoleamine-2, 3-dioxygenase inhibition has been developed as a promising approach for tumor immunotherapy, but the immunotherapy effect is limited due to effector T lymphocytes such as helper T cells (CD 4) in tumor immune escape+) And cytotoxic lymphocytes (CTLs) in the tumorThe bit has a lower wetting. Therefore, in order to enhance the clinical therapeutic effect, indoleamine-2, 3-dioxygenase (IDO) inhibition requires synergy with other therapeutic approaches that promote tumor infiltration of anti-tumor T lymphocytes, and combination therapy has become an effective approach for improving the anti-tumor efficacy, such as combination chemotherapy, photodynamic therapy or radiotherapy [ Seeber a, klinglair G, Fritz J, et al.]、[Xing L,Gong J H,Wang Y,et al.Hypoxia alleviation-triggered enhanced photodynamic therapy in combination with IDO inhibitor for preferable cancer therapy.Biomaterials 2019,206,170-182.]、[Liu M,Li Z,Yao W,et al.IDO inhibitor synergized with radiotherapy to delay tumor growth by reversing T cell exhaustion.MOL.MED.REP.2020,21,445-453.]。
Nitric Oxide (NO) plays an important role in the formation and progression of tumors as a multifunctional signaling molecule. Nitric oxide not only can induce apoptosis of various tumors through mitochondria and DNA damage, but also can regulate tumor immune microenvironment. However, the synergistic effect of nitric oxide and indoleamine-2, 3-dioxygenase inhibition on tumor immunotherapy has not been studied.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a glutathione-sensitive nano drug delivery system and a preparation method and application thereof. The invention adopts a nano metal organic framework compound Cu-BTC sensitive to glutathione as a nano carrier to mediate nitric oxide and micromolecule indoleamine-2, 3-dioxygenase inhibitor to cooperate with immunotherapy of tumor, targets micromolecule drugs to a tumor area, and enhances the immunotherapy effect of tumor.
The invention aims to provide a glutathione sensitive nano drug delivery system.
The invention also aims to provide a preparation method of the nano drug delivery system.
The invention further aims to provide application of the nano drug delivery system in preparation of anti-tumor drugs.
The above purpose of the invention is realized by the following technical scheme:
the invention firstly provides a glutathione-sensitive nano drug delivery system, which takes a metal organic framework compound Cu-BTC as a carrier and encapsulates indoleamine 2, 3-dioxygenase inhibitor and nitric oxide donor.
The nano drug delivery system provided by the invention adopts a Glutathione (GSH) -sensitive metal organic framework compound (MOF) to encapsulate an immunosuppressive enzyme indoleamine 2, 3-dioxygenase (IDO) inhibitor and a Nitric Oxide (NO) donor, has higher T-1 relaxivity, and can be used for in vivo Magnetic Resonance Imaging (MRI) visualized drug delivery. When the nano drug delivery system reaches tumor cells, high-concentration glutathione (0.5-10 mM) in the cells and an MOF metal organic framework generate a cascade reaction, divalent copper ions in the Cu-BTC can generate an oxidation reduction reaction with the glutathione to be converted into monovalent copper ions, the coordination effect inside the Cu-BTC is weakened, the metal organic framework nano carrier Cu-BTC is disintegrated, small molecule indoleamine-2, 3-dioxygenase inhibitor and a nitric oxide donor in holes are rapidly released, and then the nitric oxide donor and the monovalent copper ions react to generate a large amount of NO gas. The released small molecule indoleamine-2, 3-dioxygenase inhibitor can inhibit the activity of indoleamine-2, 3-dioxygenase and relieve the immunosuppression of anti-tumor T lymphocytes, and NO generated in situ can further reshape the microenvironment for tumor immunosuppression. Indoleamine-2, 3-dioxygenase (IDO) inhibitor and Nitric Oxide (NO) gas synergistically regulate an immunosuppressive tumor microenvironment, remarkably increase cytotoxic T lymphocyte infiltration, and reduce the number of regulatory T cells (Tregs), thereby realizing efficient immunotherapy.
It is known that small molecule drugs often lack tumor specificity, which limits their therapeutic applications and easily causes side effects in vivo, which makes efficient in vivo delivery of small molecule drugs a hotspot of current research. On the other hand, combination therapy generally requires effective co-delivery of different small molecule drugs in a spatio-temporally controlled manner. That is, in combination therapy, not only tumor-targeted delivery of drugs is very important, but also release of different drugs at the site of action at a specific site of a tumor is critical. The nano drug-loaded system provided by the invention can be accumulated at a specific tumor site, and meanwhile, different drugs coated by programming can be released by introducing a well-designed stimulus response structure into a nano carrier, so that different action sites can be accurately targeted. The nano drug delivery system can effectively mediate nitric oxide and a small molecule indoleamine-2, 3-dioxygenase inhibitor to exert an immune combined treatment effect, enhance the anti-tumor immune activity and simultaneously reduce the off-target immune toxicity of the immune regulation drug to the maximum extent.
Preferably, the indoleamine 2, 3-dioxygenase inhibitor includes, but is not limited to, any one of BMS-986205, INCB24360, NLG919, 1-methyl-D-tryptophan.
In a preferred embodiment of the invention, the indoleamine 2, 3-dioxygenase inhibitor is BMS-986205.
Preferably, the nitric oxide donor includes, but is not limited to, any one of s-nitrosothiol, organic nitrite, N-azoenediol compounds, nitrobenzene.
In a preferred embodiment of the invention, the nitric oxide donor is s-nitrosothiol.
The metal organic framework compound Cu-BTC is prepared by adopting the following method as a preferable implementation mode:
4.7g of copper nitrate trihydrate is dissolved in 200mL of methanol; dissolving 2.58g of m-benzenetricarboxylic acid in 200mL of methanol; dropwise adding the methanol solution of the m-benzenetricarboxylic acid into the methanol solution of the copper nitrate trihydrate at the speed of 100-150 mu L/min, and stirring for 2-8 h at room temperature; centrifuging at 10000rpm for 5min, collecting blue precipitate, and washing with N, N-dimethylformamide solution for several times; and finally, drying for 8-12 h in a vacuum oven at 110-150 ℃ to obtain Cu-BTC powder.
As a preferable embodiment, the preparation method of the above drug delivery nano-system of the present invention comprises the following steps:
s1, dissolving indoleamine-2, 3-dioxygenase inhibitor, nitric oxide donor and metal organic framework compound Cu-BTC in dimethyl sulfoxide, uniformly mixing for the first time, adding polyether F127, and uniformly mixing for the second time;
s2, dropwise adding the solution prepared in the step S1 into deionized water at the speed of 100-150 mu L/min, standing for 2-8 h, and carrying out vacuum evaporation, dialysis and freeze drying to obtain the nano drug-loading system.
Preferably, in the step S1, the dosage ratio of the indoleamine-2, 3-dioxygenase inhibitor, the nitric oxide donor, the metal-organic framework compound Cu-BTC, dimethyl sulfoxide, and polyether F127 is 3-6 mg: 3-6 mg: 10-30 mg: 1-3 mL: 60-120 mg.
Preferably, the first mixing in the step S1 is carried out for 0.5-2 hours at room temperature; and the second time of mixing is stirring for 2-4 hours at room temperature.
Preferably, the dialysis in step S2 is to remove the organic solvent and the free drug by dialysis in a cellulose dialysis bag with a cut-off molecular weight MWCO of 3500Da for 48-96 h. Dialysis is preferably carried out for 72 h.
Preferably, the temperature of the freeze drying in the step S2 is-50 ℃ to-10 ℃, and the time is 4-8 h.
The method of the invention modifies F127(Polyethylene-polypropylene glycol) on the surface of BMS-SNAP @ Cu-BTC nano material which encapsulates indoleamine 2, 3-dioxygenase inhibitor BMS-986205 and nitric oxide donor s-nitrosothiol in Cu-BTC so as to improve the water dispersibility and biocompatibility of the nano drug delivery system BMS-SNAP-MOF.
The nano drug delivery system BMS-SNAP-MOF is used for treating the Triple Negative Breast Cancer (TNBC) of the mouse, the tumor of the mouse is obviously regressed, and the excellent tumor immunotherapy effect is shown.
Therefore, the application of the nano drug delivery system or the nano drug delivery system BMS-SNAP-MOF prepared by the method in preparing the anti-tumor drug is also within the protection scope of the invention.
The nano drug delivery system BMS-SNAP-MOF can play an immune treatment effect on various solid tumors such as malignant melanoma, lung cancer, cervical cancer, breast cancer and bladder cancer. In a preferred embodiment of the invention, the tumor is Triple Negative Breast Cancer (TNBC).
The invention has the beneficial effects that:
the invention provides a glutathione-sensitive nano drug delivery system BMS-SNAP-MOF, wherein a metal organic framework compound Cu-BTC is adopted as a nano carrier to mediate nitric oxide and micromolecular indoleamine-2, 3-dioxygenase inhibitor for immune combined treatment of tumors, the indoleamine-2, 3-dioxygenase inhibitor and nitric oxide synergistically regulate an immune suppression tumor microenvironment, and T cells are obviously increased and regulated T cells are reduced, so that a high-efficiency tumor immune treatment effect is realized, and the problem of limited immune treatment effect of singly using the indoleamine-2, 3-dioxygenase inhibitor in the prior art is solved.
The preparation method of the nano drug delivery system BMS-SNAP-MOF is simple, is suitable for large-scale popularization, and provides a novel and efficient drug scheme for immunotherapy of solid tumors.
Drawings
FIG. 1 is a flow chart of nano drug delivery system BMS-SNAP-MOF preparation;
FIG. 2 is a metal organic framework Cu-BTC XRD X-ray diffraction pattern;
FIG. 3 is a metal organic framework Cu-BTC XPS X-ray photoelectron spectroscopy;
FIG. 4 shows the adsorption desorption of a metal organic framework Cu-BTC N2;
FIG. 5 is a distribution curve of pore sizes of a Cu-BTC metal organic framework;
FIG. 6 is a transmission electron microscope image of a metal organic nanocarrier BMS-SNAP-MOF TEM;
FIG. 7 is fluorescence imaging of intracellular nitric oxide production;
FIG. 8 is a flow-based quantitative analysis of the intracellular nitric oxide content;
FIG. 9 is magnetic resonance imaging of a tumor site in a mouse;
FIG. 10 is a flow assay of immune Cytotoxic T Lymphocytes (CTL).
Detailed Description
The invention will be further described with reference to the drawings and the detailed description, which are not intended to limit the invention in any way. The starting reagents employed in the examples of the present invention are, unless otherwise specified, those that are conventionally purchased.
Laser confocal fluorescence microscopy (germany zeiss LSM 510);
flow cytometric analyzers were purchased from Becton Dickinson (facscan to II) usa;
cell counters were purchased from american merck (Scepter 2.0);
mouse breast cancer cells (4T1) were purchased from the shanghai cell bank of the chinese academy of sciences.
Example 1
FIG. 1 is a flow chart for the preparation of BMS-SNAP-MOF.
Preparation and characterization of first, Cu-BTC
1. Preparation of
4.7g of copper nitrate trihydrate is dissolved in 200mL of methanol; dissolving 2.58g of m-benzenetricarboxylic acid in 200mL of methanol; then, dropwise adding the methanol solution of the m-benzene tricarboxylic acid into the methanol solution of the copper nitrate trihydrate at the concentration of 130 mu L/min, and stirring for 4 hours at room temperature; centrifuging at 10000rpm for 5min, collecting blue precipitate, and washing with N, N-dimethylformamide solution for several times; finally, drying is carried out in a vacuum oven at 120 ℃ for 12h, so as to obtain Cu-BTC powder (Cu-BTC MOF).
2. Characterization of
(1) The X-ray diffraction (XRD) results of Cu-BTC are shown in figure 2, and the diffraction peaks at 6.8 ° (200), 9.5 ° (220) and 11.6 ° (222) are consistent with the XRD results of Cu-BTC reported in the literature, which shows that Cu-BTC is successfully prepared, and the prepared sample has higher purity.
(2) X-ray photoelectron spectroscopy (XPS) analysis showed that Cu-BTC consists of Cu, O and C (FIG. 3).
(3)N2The results of desorption/adsorption test and pore analysis are shown in FIG. 4 and FIG. 5, respectively, and the specific surface area of the Cu-BTC powder is 1161.75m2 g-1The average pore diameter is 2.02nm, and the result shows that the prepared Cu-BTC has a mesoporous structure and potential drug loading performance.
Preparation and characterization of BMS-SNAP-MOF
1. Preparation of
5mg indoleamine-2, 3-dioxygenase inhibitor BMS-986205, 5mg nitric oxide donor s-nitrosothiol (SNAP) and 20mg Cu-BTC MOF material were first dissolved in dimethyl sulfoxide (DMSO, 2mL) and stirred ultrasonically at room temperature for 0.5h to give BMS-SNAP @ Cu-BTC. Then 100mg of polyether F127 were added to the solution and stirred at room temperature for 2 h. Dropping into deionized water at the speed of 130 mul/min, standing for 2h, and evaporating the organic solvent in vacuum. Dialyzing in cellulose bag (MWCO ═ 3500Da) for 72h, removing organic solvent and free drug, and freeze-drying at-30 deg.C for 6h to obtain BMS-SNAP-MOF.
2. Characterization of
Transmission Electron Microscopy (TEM) results of BMS-SNAP-MOF As shown in FIG. 6, the prepared BMS-SNAP-MOF had a spherical morphology and showed good dispersibility with an average size of 75 nm.
And thirdly, preparing the BMS-MOF and the SNAP-MOF by the same method as the BMS-SNAP-MOF.
Example 2 in vitro nitric oxide assay
1. Experimental methods
(1) Preparation of BMS-SNAP-MOF solution (1 mg/mL): 1mg of BMS-SNAP-MOF prepared in example 1 was dispersed in 1mL of deionized water pH7.4 and then sterile filtered with a 0.22 μm sterile filter.
(2) Cell culture
Mouse mammary cancer cells (4T1) were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ in 5% CO2Is cultured in a humid environment. Cell density was measured using a cell counter prior to the experiment.
(3) Cell processing
Cultured mouse breast cancer cells (4T1) were co-incubated with BMS-SNAP-MOF (1mg/mL), glutathione (5. mu.M) in a cell incubator for 8 h. Then, the cell culture solution was removed, 1mL of diluted 3-amino, 4-aminomethyl-2 ', 7' -diflourescin, diacetate ester (DAF-FM DA) 5. mu.M was added to cover the cells sufficiently, and the cells were incubated in a cell incubator at 37 ℃ for 20 minutes. The cells were washed three times with phosphate buffered saline (PBS, pH7.4) to sufficiently remove DAF-FM DA that had not entered the cells. And detecting the distribution condition of the cell fluorescence by using a laser confocal fluorescence microscope and a flow cytometry.
Wherein the 3-amino group, the 4-aminomethyl-2 ', 7' -bifluorine and the diacetate ester group (DAF-FM DA) are fluorescent probes for the quantitative detection of nitric oxide, and the DAF-FM DA can pass through cell membranes and can be catalyzed by esterase in cells to form DAF-FM which cannot pass through the cell membranes. DAF-FM by itself has only weak fluorescence, but can produce strong fluorescence after reaction with nitric oxide, with excitation wavelength of 495nm and emission wavelength of 515 nm. DAPI, 4', 6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole), is a fluorescent dye that binds strongly to DNA and is commonly used for fluorescence microscopy imaging.
2. Results of the experiment
The fluorescence imaging result of the intracellular nitric oxide detection is shown in fig. 7, fig. 8 is the flow quantitative analysis result of the intracellular nitric oxide content, and as can be seen from fig. 7 and 8, after mouse 4T1 breast cancer cells are treated with different nano-drugs for 8h, only 4T1 cells co-treated with BMS-SNAP-MOF and glutathione show a significant fluorescence signal, which indicates that nitric oxide gas is generated in the cells. Furthermore, BMS-SNAP-MOF was decomposed and nitric oxide gas was produced after addition of glutathione GSH in 1640 medium, confirming that it was a successful NO donor in cells. It was demonstrated that divalent copper ions in BMS-SNAP-MOF can undergo redox reaction with GSH at high concentration in the tumor microenvironment to convert to monovalent copper ions, causing the metal-organic framework to disintegrate, releasing BMS-986205 and the nitric oxide donor s-nitrosothiol SNAP in the pores. Subsequently, SNAP reacts with monovalent copper ions to produce NO gas.
Example 3 in vivo MR imaging in animal models
1. Experimental methods
BALB/c mice (4-6 weeks, 20g) were purchased from Guangdong provincial medical laboratory animal center and 4T1 cells (1.0X 10)6Cells) were suspended in 100 μ L phosphate buffered saline and injected subcutaneously into the mouse flanks to establish an animal tumor model (triple negative breast cancer). When the tumor reaches about 100mm3In time, animals were used for the experiments.
BMS-SNAP-MOF (10mg/kg) prepared in example 1 was injected into the tail vein of tumor-bearing mice, and Magnetic Resonance Imaging (MRI) studies were performed on tumor sites of the mice at 4h, 8h, 12h and 24h before and after injection, respectively.
Cu2+Is a T1 contrast agent in Magnetic Resonance Imaging (MRI), and therefore contains Cu2+The BMS-SNAP-MOF can be used as an effective T1 contrast agent in magnetic resonance imaging. T1 weighted magnetic resonance imaging of BMS-SNAP-MOF was evaluated using a 3.0T MRI clinical scanner. Magnetic resonance imaging can evaluate semi-quantitative biodistribution analysis of BMS-SNAP-MOF nanoparticles in 4T1 tumor-bearing mice.
2. Results of the experiment
FIG. 9 is a graph of T1 weighted NMR imaging of 4T1 tumor-bearing mice before and after intravenous tail injection of BMS-SNAP-MOF, and it can be seen from FIG. 9 that the MRI signal intensity of the tumor sites of the mice is continuously enhanced after the injection of BMS-SNAP-MOF, and the tumor sites of the mice reach the strongest level at 8h after the injection and still maintain the higher MRI signal level at 24 h. Experimental results show that after BMS-SNAP-MOF is injected into the tail of a vein, the nano-drug can be effectively accumulated at a tumor part. In addition, the excellent nuclear magnetic resonance imaging accurately defines the tumor area, and provides effective guidance for subsequent synergistic IDO/NO immunotherapy.
Example 4 in vivo Cytotoxic T Lymphocyte (CTL) assay
1. Experimental methods
BALB/c mice (4-6 weeks, 20g) were purchased from Guangdong provincial medical laboratory animal center and 4T1 cells (1.0X 10)6Cells) were suspended in 100 μ L phosphate buffered saline and injected subcutaneously into the mouse flanks to establish an animal tumor model (triple negative breast cancer). When the tumor reaches about 100mm3In time, animals were used for the experiments.
4T1 tumor-bearing mice were randomly divided into 4 groups of 5 mice each. Four groups of mice were injected tail-intravenously with PBS, BMS-MOF (10mg/kg), SNAP-MOF (10mg/kg) and BMS-SNAP-MOF (10mg/kg) prepared in example 1, every two days. On day 14, the tumor tissue is taken out and cut into small pieces, then the small pieces are ground by a tissue grinder, the cell suspension is centrifuged for 4-5min under the conditions of 2-8 ℃ and 400g of 300-. Then, the surface was stained with a fluorescent-labeled surface antibody (CD3, CD4, and CD8) at 4 ℃ for 30 minutes in the dark. After staining was completed, the cells were washed 3 times with flow cytostaining buffer and centrifuged at 1500rpm for 5 min. Finally, the cells were resuspended in 0.5mL FACS buffer, stained cells were detected using flow cytometry, and tumor immunoreactions were evaluated for different treatment groups.
2. Results of the experiment
FIG. 10 shows the flow measurement results of immunocytotoxic T lymphocytes (CTL), and from FIG. 10, it can be seen that BMS-MOF or SNAP-MOF treated group alone only partially increased the number of cytotoxic T lymphocytes at the tumor site, 21.1% and 15.7%, respectively, compared to the phosphate buffered saline treated group (8.1%). Whereas BMS-SNAP-MOF treated tumor tissue is CD8+T cell (CD 3)+CD4-CD8+) The increase of the amount is 31.4% compared with the control group. As can be seen from the results, the BMS-SNAP-MOF-treated group significantly enhanced CD8 compared to the other three groups+Infiltration of T cells at the tumor site.
The experiments show that in the BMS-SNAP-MOF nano drug delivery system, indoleamine-2, 3-dioxygenase (IDO) inhibitor and Nitric Oxide (NO) gas synergistically regulate a tumor immunosuppressive microenvironment, the content of cytotoxic T lymphocytes is remarkably increased, the number of regulatory T cells is reduced, and therefore efficient synergistic immunotherapy is achieved. In animal experiments, the nano diagnosis and treatment system shows an excellent effect on tumor immunotherapy when used for treating mouse Triple Negative Breast Cancer (TNBC).
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. The glutathione-sensitive nano drug delivery system is characterized in that a metal organic framework compound Cu-BTC is used as a carrier, and an indoleamine 2, 3-dioxygenase inhibitor and a nitric oxide donor are encapsulated.
2. The nanopharmaceutical system of claim 1, wherein the indoleamine 2, 3-dioxygenase inhibitor comprises any of BMS-986205, INCB24360, NLG919, 1-methyl-D-tryptophan.
3. The nanoplatelets system of claim 1 wherein the nitric oxide donor comprises one or more of s-nitrosothiol, organic nitrite, N-azoenediol compounds, nitrobenzene.
4. The preparation method of the nano drug delivery system of any one of claims 1 to 3, which is characterized by comprising the following steps:
s1, dissolving indoleamine-2, 3-dioxygenase inhibitor, nitric oxide donor and metal organic framework compound Cu-BTC in dimethyl sulfoxide, uniformly mixing for the first time, adding polyether F127, and uniformly mixing for the second time;
s2, dropwise adding the solution prepared in the step S1 into deionized water at the speed of 100-150 mu L/min, standing for 2-8 h, and carrying out vacuum evaporation, dialysis and freeze drying to obtain the nano drug-loading system.
5. The method according to claim 4, wherein the indoleamine-2, 3-dioxygenase inhibitor, the nitric oxide donor, the metal-organic framework compound Cu-BTC, the dimethyl sulfoxide and the polyether F127 are used in an amount ratio of 3-6 mg in step S1: 3-6 mg: 10-30 mg: 1-3 mL: 60-120 mg.
6. The method according to claim 4, wherein the first blending in step S1 is performed for 0.5-2 hours at room temperature; and the second time of mixing is stirring for 2-4 hours at room temperature.
7. The method of claim 4, wherein the dialysis in step S2 is carried out in a cellulose dialysis bag with a cut-off Molecular Weight (MWCO) of 3500Da for 48-96 h to remove organic solvent and free drug.
8. The method of claim 4, wherein the temperature of the freeze-drying in step S2 is-50 ℃ to-10 ℃ for 4-8 hours.
9. The nano drug delivery system of any one of claims 1 to 3 or the nano drug delivery system prepared by the method of any one of claims 4 to 7, for use in the preparation of an anti-tumor drug.
10. The use of claim 9, wherein the tumor is a triple negative breast cancer.
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