Layered double-metal hydroxide responsive nano-drug modified by lactate oxidase as well as preparation and application of layered double-metal hydroxide responsive nano-drug

Abstract

The invention relates to a lactate oxidase modified layered double hydroxide responsive nano-drug as well as preparation and application thereof. The composite material comprises a copper ion doped LDH (layered double hydroxide) nano-material, a drug and lactate oxidase. The nano-drug has the effects of regulating lactic acid metabolism and oxidation-reduction metabolism of tumors, increasing secretion of immune factors, enhancing chemical kinetics therapy and realizing immunotherapy and chemical kinetics combined therapy, and a new thought is provided for constructing the safe, efficient and multifunctional nano-drug.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to a lactate oxidase-modified layered double hydroxide responsive nanomedicine and a preparation method and application thereof. Background Art

[0002] Cancer remains a major public health problem worldwide, with high morbidity and mortality. With the progress of tumor biology research, cancer is now considered to be a comprehensive disease that interacts with gene mutations, immune deficiency and metabolic disorders. Metabolic reprogramming is one of the important hallmarks of cancer. Therefore, targeting the tumor microenvironment (TME) to change tumor metabolic pathways is considered to be an ideal strategy for the next generation of cancer treatment.

[0003] The special metabolic pathways of tumor cells and the accumulation of metabolites in the TME greatly limit the immune monitoring and killing ability of effector cells in the immune system against tumor cells. More and more studies have shown that lactic acid accumulated in the tumor microenvironment not only promotes the growth, invasion and metastasis of the tumor itself, but also impairs the function of effector T cells and natural killer cells, promotes the repolarization of macrophages to M2, thereby forming an immunosuppressive microenvironment and inactivating the immune system (Cell Metabolism, 2023, 35, 1101-1113). Fortunately, some strategies targeting lactate metabolism have been developed to activate the immune system and achieve immunotherapy. For example, small molecule inhibitors are used to inhibit the efflux channel of lactate (monocarboxylate transporter), reduce the efflux of lactate in tumor cells, and thus reduce the lactate content in the tumor microenvironment, which can activate immunotherapy to a certain extent (Cell Reports, 2019, 29, 135–150). However, the disadvantages of small molecule drugs such as easy metabolism and low bioavailability limit their therapeutic effects. With the development of nanomedicine, strategies that use nanocarriers to load lactate oxidase to regulate lactate metabolism and combine it with sonodynamic therapy, chemotherapy or mild photothermal therapy have been reported and have shown good anti-tumor effects (ACS Nano, 2021, 15, 16934-16945). Based on the existing research progress, it can be concluded that lactate can be considered a universal metabolic target, and reducing the lactate content in the tumor microenvironment can activate immunotherapy and achieve immune monitoring. However, tumor cell metabolism is plastic, and most of the combined therapy strategies reported so far are lactate oxidase consumption of lactate (ACS Nano, 2021, 15, 16934-16945), or inhibitors inhibit lactate efflux (Advanced Materials, 2023, 36, 2308774), which have limited regulation of lactate metabolism. Therefore, designing a nanoplatform that can simultaneously consume lactate in the tumor microenvironment, inhibit lactate efflux in tumor cells, and combine other metabolic regulation is expected to provide a new strategy for tumor treatment.

[0004] In recent years, the redox metabolism of tumor cells has been considered an effective target for tumor treatment. Chemodynamic therapy (CDT), developed based on the high content of hydrogen peroxide (H2O2) in the tumor microenvironment and the Fenton / Fenton-like reaction mediated by metal ions, is considered to be a typical representative of tumor treatment by changing tumor redox metabolism (Nano Today, 2020, 35, 100946). The metal ion-mediated Fenton / Fenton-like reaction converts H2O2 into highly toxic hydroxyl radicals (·OH), which further cause cells to produce lipid peroxides, thereby inducing cell apoptosis and necrosis. This treatment method has been widely explored due to its low invasiveness and high tumor specificity. Although CDT has great potential in tumor treatment, its therapeutic effect still faces many limitations: 1) The content of H2O2 in the tumor is not enough to continuously produce ·OH; 2) The highly expressed reducing substances (such as glutathione (GSH)) in the tumor microenvironment (TME) weaken the therapeutic effect of CDT. These limitations make it difficult to completely eliminate malignant tumors by CDT alone. Therefore, it is crucial for efficient tumor treatment to rationally construct nanocarrier systems to load chemodynamic therapeutic agents and reprogram the tumor microenvironment, such as increasing the H2O2 concentration at the tumor site, thereby enhancing the CDT effect.

[0005] A search of relevant domestic and foreign literature and patents shows that there is no report on using copper-doped LDH as a carrier to load diclofenac (DC) (a monocarboxylate transporter inhibitor that inhibits lactate efflux) and modify lactate oxidase (LOX) for regulating tumor metabolism to activate immunotherapy and enhance chemodynamic therapy. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a lactate oxidase-modified layered double hydroxide responsive nanomedicine and its preparation and application.

[0007] The invention provides a layered double metal hydroxide (LDH) composite material, which comprises a copper ion-doped LDH nanomaterial, a drug and lactate oxidase; wherein the copper ion-doped LDH nanomaterial is used as a carrier to load the drug and modify the lactate oxidase.

[0008] Preferably, the drug includes diclofenac DC; and the copper ion-doped LDH nanomaterial is copper ion-doped LDH nanoparticles.

[0009] Preferably, the mass ratio of the copper ion-doped LDH nanomaterial to lactate oxidase is 0.9-1.1:0.01-0.03.

[0010] The present invention provides a method for preparing any of the above-mentioned layered double hydroxide LDH composite materials, comprising:

[0011] (1) mixing the drug, magnesium salt, copper salt, aluminum salt and water, adjusting the pH value, stirring the reaction under oil bath conditions, centrifuging, washing and freeze-drying to obtain a drug-loaded copper ion-doped LDH nanomaterial;

[0012] (2) Lactate oxidase LOX, drug-loaded copper ion-doped LDH nanomaterials, and a solvent are mixed, stirred for reaction, centrifuged, and washed to obtain a layered double hydroxide LDH composite material.

[0013] Preferably, in step (1), the molar ratio of the drug, magnesium salt, copper salt and aluminum salt is 0.035-0.055:0.050-0.070:0.02-0.04:0.035-0.055;

[0014] Preferably, the drug in step (1) includes diclofenac DC; the magnesium salt includes MgCl2·6H2O; the copper salt includes CuCl2·2H2O; and the aluminum salt includes AlCl3·6H2O.

[0015] Preferably, the drug in step (1) is a drug solution; wherein the solvent of the drug solution is an aqueous solution containing 8-10% (volume) dimethyl sulfoxide (DMSO);

[0016] Preferably, in step (1), the pH value is adjusted to 9.0-10.0, and a base is used to adjust the pH value, and more preferably the pH value is 9.3-9.7; wherein the base is 1M NaOH solution.

[0017] Preferably, the stirring reaction under oil bath conditions in step (1) is carried out at 40-60° C. in the oil bath, in the dark, under nitrogen protection, and the stirring reaction time is 24-48 h.

[0018] The whole process of preparing the drug-loaded copper ion-doped LDH nanomaterial in step (1) is carried out in an oil bath at 50° C., in the dark and under nitrogen protection, and the stirring reaction time is 24 to 48 hours.

[0019] Preferably, in step (2), the mass ratio of LDH doped with copper ions to lactate oxidase in the drug-loaded LDH nanomaterial doped with copper ions is 0.9-1.1:0.01-0.03.

[0020] Preferably, the solvent in step (2) is water, and further preferably ultrapure water; the stirring reaction is carried out at room temperature for 2 to 6 hours, and further preferably the stirring reaction time is 3 to 5 hours.

[0021] Furthermore, the entire process of step (2) is carried out at room temperature.

[0022] Furthermore, the layered double hydroxide LDH composite material in step (2) is a lactate oxidase-modified LDH-responsive nanodrug LDH-DC-LOX.

[0023] The present invention provides an application of any layered double metal hydroxide (LDH) composite material in the preparation of tumor nanomedicines for metabolic regulation and chemokinetics-immunotherapy combined therapy.

[0024] The present invention provides an application of any layered double metal hydroxide (LDH) composite material in preparing a drug for treating breast cancer.

[0025] The present invention utilizes a coprecipitation method to synthesize a pH-responsive LDH-DC nanodrug doped with copper ions and loaded with DC, and then utilizes electrostatic interaction to modify LOX on the surface of the nanodrug to obtain a pH-responsive nanodrug LDH-DC-LOX modified with lactate oxidase, which is used to regulate tumor metabolism and achieve combined immunotherapy / chemodynamic therapy.

[0026] The present invention integrates copper ions, DC and lactate oxidase with LDH, which can degrade the lactic acid content in the tumor microenvironment and inhibit the lactic acid efflux in the tumor, thereby promoting lactate oxidase to convert lactic acid into pyruvate and H2O2, activating immunotherapy on the one hand and enhancing chemodynamic therapy on the other hand, thereby achieving immunotherapy / chemodynamic therapy combined treatment.

[0027] The present invention uses Zeta potential and dynamic light scattering analysis (DLS), ultraviolet visible absorption spectrum (UV-vis), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and other means to characterize the physical and chemical properties of the prepared lactate oxidase-modified copper ion-doped and DC-loaded LDH-responsive nanodrug. Then, the CCK-8 method was used to analyze and evaluate the cytotoxicity of LDH-DC-LOX and related control materials; the lactate detection kit was used to evaluate the lactate levels inside and outside the cells after treatment with different materials, and the GSH detection kit was used to evaluate the GSH levels after treatment with different materials; the laser confocal microscope was used to evaluate the effects of the materials on intracellular ROS and LPO levels; a subcutaneous breast cancer (4T1) model of white mice was established for in vivo anti-tumor experiments and the tumor masses of the mice after treatment were analyzed for immune analysis; a subcutaneous 4T1 model of white mice was established to inject nanodrugs into the tumor and collect the urine and feces of the mice to analyze the metabolic pathways of the nanodrugs in the body; the LDH-DC-LOX nanodrug was injected into the tail vein of healthy white mice, and the blood routine and blood biochemical indicators of the mice were tested to evaluate the biosafety of the nanodrug.

[0028] The lactate oxidase-modified LDH nanomedicine in the present invention can regulate the universal metabolic target "lactate" to activate immunotherapy, increase the secretion of immune factors, enhance chemodynamic therapy, and achieve combined immunotherapy and chemodynamic therapy, providing a new idea for the construction of safe, efficient and multifunctional nanomedicines.

[0029] Beneficial Effects

[0030] (1) The synthesis process of the present invention is simple, the reaction conditions are mild, the raw materials are abundant and the cost is low, and it has good development prospects;

[0031] (2) The nanomedicine prepared by the present invention has the function of degrading intracellular and extracellular lactic acid, effectively regulating the lactic acid metabolic pathway in tumors, activating the body's immunotherapy, and providing a new idea for constructing safe, simple and efficient nanomedicines;

[0032] (3) The copper ions doped in the nanomedicine prepared by the present invention can catalyze H2O2 in the TME to generate ·OH, reduce the intracellular GSH content, change the tumor redox metabolic balance, and achieve chemodynamic therapy;

[0033] (4) The nanomedicine prepared by the present invention can achieve cascade regulation of tumor lactate metabolism and redox metabolism;

[0034] (5) The nanomedicine prepared by the present invention can be acid-responsively degraded and excreted from the body, and has good biosafety;

[0035] (6) The nanomedicine prepared by the present invention has a significant anti-tumor effect after being injected into the mouse through the tumor, and can achieve immunotherapy / chemodynamic combined treatment, which has potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the synthesis and application of LDH-DC-LOX prepared in the present invention;

[0037] Figure 2 X-ray diffraction patterns of LDH-DC prepared in Example 1 and LDH prepared in Comparative Example 1 measured by an X-ray diffractometer;

[0038] Figure 3 (A) is a TEM image of LDH prepared in Comparative Example 1, and (B) is a TEM image of LDH-DC prepared in Example 1 (scale bar is 100 nm);

[0039] Figure 4 The UV absorption spectra of DC, LOX, LDH-DC and LDH-DC-LOX prepared in Example 1, and LDH prepared in Comparative Example 1;

[0040] Figure 5 The infrared absorption spectra of DC, LOX, LDH-DC and LDH-DC-LOX prepared in Example 1, and LDH prepared in Comparative Example 1;

[0041] Figure 6 The hydrated particle size (A), polydispersity coefficient (B), and surface potential (C) of LDH-DC-LOX prepared in Example 1, LDH and LDH-LOX prepared in Comparative Example 1, and LDH-DC-BSA prepared in Comparative Example 2;

[0042] Figure 7 It is a SDS-polyacrylamide gel electrophoresis (SDS-PAGE) diagram of LOX, BSA, LDH-DC and LDH-DC-LOX prepared in Example 1, and LDH-DC-BSA prepared in Comparative Example 2;

[0043] Figure 8 Cu is the Cu of LDH-DC-LOX prepared in Example 1 at different pH values 2+ Release kinetics curve (A) and DC release kinetics curve (B);

[0044] Fig. 9 The changes in lactic acid content after incubation of LOX, LDH-DC-LOX prepared in Example 1, LDH-LOX prepared in Comparative Example 1, and LDH-DC-BSA prepared in Comparative Example 2 with lactic acid for different time periods;

[0045] Fig.10 MB degradation by LDH-DC-LOX prepared in Example 1 under different conditions;

[0046] Fig.11 The ESR spectra of LDH-DC-LOX prepared in Example 1 under different conditions;

[0047] Fig.12 The cell viability of 4T1 cells after being treated with different concentrations of LDH-DC-LOX prepared in Example 1, LDH and LDH-LOX prepared in Comparative Example 1, and LDH-DC-BSA prepared in Comparative Example 2 (A); and the cell viability of 4T1 cells after being treated with different materials when the LDH concentration is 10 μg / mL (B);

[0048] Fig.13 Extracellular lactate content (A) and intracellular lactate content (B) after 4T1 cells were incubated with DC, LOX, LDH, LDH-DC-BSA, LDH-LOX or LDH-DC-LOX for 12 h;

[0049] Fig.14is the intracellular GSH content after 4T1 cells were incubated with DC, LOX, LDH, LDH-DC-BSA, LDH-LOX or LDH-DC-LOX for 12 h;

[0050] Fig.15 Flow cytometry histogram (A) and quantitative analysis (B) of intracellular ROS levels detected after 4T1 cells were incubated with DC, LOX, LDH, LDH-DC-BSA, LDH-LOX or LDH-DC-LOX for 6 h;

[0051] Fig.16 Laser confocal images of 4T1 cells detected intracellular ROS levels after incubation with DC, LOX, LDH, LDH-DC-BSA, LDH-LOX or LDH-DC-LOX for 6 h (scale bar: 20 μm);

[0052] Fig.17 Laser confocal images (A, scale bar 20 μm) and quantitative analysis (B) of 4T1 cells detected the intracellular LPO level after incubation with DC, LOX, LDH, LDH-DC-BSA, LDH-LOX or LDH-DC-LOX for 6 h;

[0053] Fig.18 Flow cytometry dot plots (A) and quantitative analysis (B) of apoptosis effects detected after 4T1 cells were incubated with DC, LOX, LDH, LDH-DC-BSA, LDH-LOX or LDH-DC-LOX for 24 h;

[0054] Fig.19 Figure 17 shows the relative weight change of mice in each group after different treatments within 14 days (A), the relative tumor volume change of mice (B), and the tumor mass (C);

[0055] Fig. 20 The changes in lactic acid content in tumor tissues of each group of mice after different treatments in Example 17;

[0056] Fig.21 H&E and TUNEL staining of tumor tissue and H&E staining of lung tissue of each group of mice after different treatments in Example 17 (scale bar: 50 μm);

[0057] Fig. 22 The apoptosis rate of tumor cells was quantitatively analyzed by TUNEL staining of tumor tissues of mice in each group in Example 17;

[0058] Fig.23 The CD3 in the tumor of each group of mice after different treatments in Example 17 + CD4 + (A) CD3 + CD8+ (B) Flow cytometry and quantitative analysis of T cell typing (E) and CD3 + CD4 + (C) CD3 + CD8 + (D) Flow cytometry analysis and quantitative analysis of T cell typing (F);

[0059] Fig.24 The immunofluorescence staining images of CD4 T cells, CD8 T cells, M2 macrophages, M1 macrophages, and NK T cells in the tumors of each group of mice after different treatments in Example 17 (scale bar is 50 μm);

[0060] Fig.25 The immunofluorescence staining images of CD4 T cells and CD8 T cells in the spleen of each group of mice after different treatments in Example 17 (scale bar is 50 μm);

[0061] Fig.26 The contents of Granzyme B (A), IFN-γ (B), and TNF-α in the serum of each group of mice after different treatments in Example 17;

[0062] Fig. 27 Figure (A) is the Western blot test result of IRF1, SCL7A11 and GPX4 proteins in the tumors of each group of mice after different treatments in Example 17. (BD) is the grayscale quantitative graph of each protein band;

[0063] Fig.28 The changes in GSH content in tumor tissues of each group of mice after different treatments in Example 17;

[0064] Fig.29 The following are ROS and LPO staining images of tumor tissues of mice in each group after different treatments in Example 17 (A, scale bar is 50 μm), ROS quantitative analysis image (B), and LPO quantitative analysis image (C);

[0065] Fig.30 is the copper content in the urine (A), feces (B) and main tissues (C) of mice in Example 18;

[0066] Fig.31 H&E staining of the main organs (heart, liver, spleen, and kidney) of mice in each group after different treatments in Example 17 (scale bar: 50 μm);

[0067] Fig.32 (A-D) are blood routine tests after different materials were injected into the tail vein of mice in Example 19;

[0068] Fig.33 (A-D) are blood biochemical tests after different materials were injected into the tail vein of mice in Example 19. DETAILED DESCRIPTION

[0069] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0070] Unless otherwise specified, all chemical reagents were used directly without further purification. MgCl2·6H2O, CuCl2·6H2O, AlCl3·6H2O, and NaOH were purchased from Sinopharm Chemical Reagent Co., Ltd. Lactic acid (LA) was purchased from Beijing Bailingwei Technology Co., Ltd. Lactic acid detection kit was purchased from Beijing Solebao Technology Co., Ltd. Methylene blue (MB) was purchased from Sigma-Aldrich China Co., Ltd. (Shanghai, China). DC was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. 5,5-Dimethylpyrroline N-oxide (DMPO) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. 4T1 cells were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. DMEM medium, fetal bovine serum, penicillin-streptomycin double antibody, and trypsin were purchased from Hangzhou Jinuo Biomedical Technology Co., Ltd. BCAassay kit, Cell Counting Kit-8 (CCK-8), apoptosis detection kit, and ROS detection probe (DCFH-DA) were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. GSH detection kit was purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd. C11-BODIPY 581 / 591 Lipid peroxidation fluorescent probe was purchased from Shanghai Maokang Biotechnology Co., Ltd. DAPI and singlet oxygen detection kit were purchased from Beibo Biotechnology Co., Ltd. Female BALB / C mice were purchased from Shanghai Slake Laboratory Animal Center. IFN-γ cytokine ELISA detection kit, TNF-α cytokine ELISA detection kit, and Granzyme B cytokine detection kit were purchased from Hangzhou Lianke Biotechnology Co., Ltd. Anti-CD3-PE, Anti-CD8-FITC, and Anti-CD4-FITC were purchased from Wuhan Sanying Biotechnology Co., Ltd. IRF1 antibody, SLC7A11 antibody, and GPX4 antibody were purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0071] Example 1

[0072] (1) Prepare a 15 mL DC (0.045 M) solution. Add the DC solution to a 15 mL solution containing MgCl2·6H2O (0.06 M), CuCl2·6H2O (0.03 M), and AlCl3·6H2O (0.045 M). Slowly add NaOH (1 M) solution to adjust the pH of the mixed solution to 9.5 ± 0.2. Stir in an oil bath at 50 °C in a nitrogen atmosphere and away from light for 24 h. After stirring, centrifuge at 3800 g for 10 min to collect the sample, wash to remove the two free salt ions and DC, and freeze-dry the final product to obtain Cu doped 2+ LDH-DC nanoparticles (NPs) were prepared for related characterization and subsequent experiments. The solvent of the DC solution in the above steps is an aqueous solution containing 10% DMSO, and the solvents of other solutions are ultrapure water without carbon dioxide. By elemental analysis to detect the C content in LDH and LDH-DC, it can be calculated that the DC content in LDH-DC NPs is 33.3% and the LDH content is 66.7%.

[0073] (2) The LDH-DC NPs solution in step (1) was mixed with the LOX solution at a ratio of LDH:LOX=1:0.02, stirred at 37°C in the dark for 4 h, and centrifuged at 3800 g for 10 min to obtain an LDH-DC-LOX nanocomplex.

[0074] Example 2

[0075] Take an appropriate amount of solid powder of LDH-DC in Example 1 and LDH in Comparative Example 1 and use an X-ray diffractometer to detect its crystal structure. Figure 2 The X-ray crystal diffraction patterns of LDH and LDH-DC showed that the peaks of the two nanomaterials were sharp and narrow, and the peak shapes were consistent with those reported in the literature, indicating that the synthesized LDH and LDH-DC nanoparticles had good crystallinity and the loading of DC did not change the crystal structure of LDH.

[0076] Example 3

[0077] The LDH-DC in Example 1 and the LDH in Comparative Example 1 were characterized in terms of size and morphology. As shown in the TEM images, LDH ( Figure 3 A) and LDH-DC ( Figure 3 B) Both exhibit hexagonal morphology with diameters of approximately 50 nm and 55 nm, respectively.

[0078] Example 4

[0079] Appropriate amounts of LDH-DC, LDH-DC-LOX prepared in Example 1, LDH and LDH-LOX prepared in Comparative Example 1, and LDH-DC-BSA nanoparticles prepared in Comparative Example 2 were diluted with ultrapure water to prepare a solution with an LDH concentration of 0.5 mg / mL for measuring the ultraviolet absorption spectrum, hydrated particle size, and surface potential. Figure 4 As shown, LDH-DC showed a DC peak at 284nm and shifted, indicating that DC was successfully loaded on LDH nanoparticles. LDH-DC-LOX showed an absorption peak at 285nm, indicating that LOX was successfully modified onto LDH-DC nanoparticles. Infrared absorption spectrum ( Figure 5 ) shows that LDH-DC is at 744cm -1 The vibration peaks belonging to C-Cl also indicate that LDH can successfully load DC. -1 The absorption peaks that appeared came from the vibration of the C=O bond, so the infrared absorption spectrum can also prove that DC was successfully loaded onto LDH and could be further loaded with LOX. Figure 6 As shown in (A), the hydrated particle size of LDH is 109.8 nm, the hydrated particle size of LDH-DC is 132.1 nm, and after loading LOX, the hydrated particle size of LDH-DC-LOX increases to 158.3 nm. The hydrated particle size of LDH-DC-BSA prepared in Comparative Example 1 is 150.2 nm. The hydrated particle size of LDH-LOX prepared in Comparative Example 2 is 145.3 nm. Polydispersity test results ( Figure 6 B) shows that after loading drugs and LOX, LDH-DC-LOX nanomedicine still maintains good dispersibility. The surface potential test results are as follows Figure 6 (C) shows that the potential of LDH is 33.9 mV, the potential of LDH-DC is 20.8 mV, and after loading LOX, the potential of LDH-DC-LOX decreases to 15.5 mV. The potential of LDH-DC-BSA in Comparative Example 1 is 13.3 mV. The potential of LDH-LOX in Comparative Example 2 is 22.1 mV. The hydrated particle size and potential changes prove the successful synthesis of LDH-DC-LOX, LDH-DC-BSA and LDH-LOX.

[0080] Example 5

[0081] Take LDH-DC, LDH-DC-LOX prepared in Example 1, and LDH-DC-BSA prepared in Comparative Example 2 for SDS-PAGE characterization to verify the successful loading of LOX and BSA. Mix LDH-DC, LOX, LDH-DC-LOX, BSA, and LDH-DC-BSA with an appropriate amount of SDS-PAGE protein loading buffer and boil for 5 minutes to fully denature the protein. After each group of samples has cooled to room temperature, load them into the SDS-PAGE gel loading wells. Use a constant voltage method for the entire SDS-PAGE process, set the voltage to 100V, and set the time to 90 minutes. During the electrophoresis process, the electrophoresis can be stopped when bromophenol blue reaches near the bottom of the gel. Figure 7 As shown, the LDH-DC group showed no protein bands, while the LDH-DC-LOX group and the LOX group showed similar protein bands, proving that LOX was successfully loaded onto LDH-DC. Similarly, the LDH-DC-BSA group and the BSA group showed protein bands near the same molecular weight, proving that BSA was successfully loaded onto LDH-DC.

[0082] Example 7

[0083] Prepare buffer solutions of pH = 7.4 and pH = 6.5 respectively for analyzing Cu in LDH-DC 2+ and DC responsive release. The prepared LDH-DC-LOX was dissolved in 1 mL of the above-mentioned different buffer solutions into a 1 mg / mL solution and placed in a dialysis bag (molecular retention capacity of 3000 Da). The dialysis bag was then placed in a container containing 9 mL of different buffer solutions and shaken in a constant temperature shaker at 37°C. At different time points, 1 mL of the buffer solution outside the dialysis bag was aspirated, and then 1 mL of the corresponding buffer solution was added to the container, and the absorbance of the removed liquid was measured at 277 nm. In addition, the copper ion content in the removed liquid was detected by inductively coupled plasma optical emission spectrometer (ICP-OES). After the sustained release, the DC and Cu of LDH-DC-LOX under different conditions were plotted. 2+ Release curve. Figure 8 (AB) shows that LDH-DC-LOX is in a buffer solution at pH 7.4. 2+ and DC released less, with the release rates of 10.1% and 15.1% respectively at 48h, while in the buffer solution of pH=6.5, Cu 2+ The release rates of LDH-DC-LOX and DC at 48 h were 32.2% and 37.5%, respectively. This indicates that LDH-DC-LOX is pH-responsive and can release Cu in a responsive manner in the tumor microenvironment. 2+ and DC.

[0084] Example 8

[0085] The LDH-DC-LOX prepared in Example 1, the LDH-LOX prepared in Comparative Example 1, and the LDH-DC-BSA prepared in Comparative Example 2 were incubated with a 5 mM lactic acid solution at a LOX concentration of 0.2 μg / mL for 1 h, 2 h, and 4 h. The lactic acid content of the nanomaterials after incubation with lactic acid for different time periods was detected using a lactic acid detection kit. The results are shown in FIG. Fig. 9 As shown, both LDH-LOX and LDH-DC-LOX can degrade lactic acid. As the incubation time increases, the lactic acid content gradually decreases, indicating that LOX still has good activity after being loaded into the nanomaterial, and the enzyme activity is retained by more than 90%.

[0086] Example 9

[0087] The LDH-DC-LOX prepared in Example 1 was used to characterize the Cu 2+ It has the property of producing hydroxyl radicals (·OH) by a Fenton-like reaction with H2O2. Since ·OH can cause MB to degrade and fade, MB is used as an indicator of ·OH. The absorbance of MB at 665nm can be detected to indirectly reflect the degradation of MB induced by ·OH. LDH-DC-LOX containing 10μg LDH was mixed with different solutions containing 1mM H2O2 and MB (10μg / mL). The solutions were mixed evenly and incubated at room temperature for 180min, and then the changes in absorbance at 665nm were monitored by UV-vis spectroscopy. Fig.10 As shown in the figure, after incubation with H2O2, LDH-DC-LOX can significantly reduce the absorbance of MB. The reason may be that LDH-DC-LOX produces ·OH through a Fenton-like reaction to degrade MB. After adding 10mM LA, the absorbance of the mixed solution at 665 was further reduced. The reason may be that the lactate oxidase loaded by LDH-DC-LOX degraded lactate into H2O2 to produce more ·OH. In addition, after adding GSH, the absorbance of the solution was further reduced. It may be that GSH degraded Cu 2+ Reduction to Cu + The above results indicate that LDH-DC-LOX is expected to produce ·OH through Fenton-like reaction in TME, and further promote Fenton-like reaction by loading lactic acid, thereby enhancing chemokinetic therapy of tumor cells.

[0088] Example 10

[0089] Electron spin resonance spectroscopy is considered an important means of detecting reactive oxygen species. 1 mg / mL of LDH-DC-LOX was transferred to a 2 mL centrifuge tube, incubated with different solutions containing H2O2 for 60 min, and the capture agent DMPO was added to detect the electron spin resonance spectrum. The results are shown in Figure 2. Fig.11As shown in the figure, after LDH-DC-LOX was incubated with H2O2, a characteristic peak of ·OH (1:2:2:1) appeared, indicating that LDH-DC-LOX can produce reactive oxygen species after incubation with H2O2, and the amount of ·OH produced can be further increased in the presence of LA and GSH. These results show that LDH-DC-LOX can interact with H2O2 to produce ·OH through a Fenton-like reaction, and in the presence of lactic acid, the loaded LOX can degrade lactic acid and increase the amount of H2O2, while GSH can promote Cu 2+ Converted to Cu + This ultimately further enhances the Fenton-like reaction and increases the amount of ·OH.

[0090] Embodiment 11

[0091] 4T1 cells were used as a model to detect the cytotoxicity of LDH-DC-LOX prepared in Example 1, LDH and LDH-LOX prepared in Comparative Example 1, and LDH-DC-BSA prepared in Comparative Example 2. 4T1 cells in the logarithmic growth phase were collected and 1×10 4 The cells were seeded in a 96-well plate at a density of 100 U / mL penicillin, 100 U / mL streptomycin and 10% FBS in a complete DMEM medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin and 10% FBS, and incubated at 5% CO2 and 37°C for 24 h. The original medium was discarded, and a medium containing LDH, LDH-DC-LOX, LDH-LOX, LDH-DC-BSA, LOX (0.2 μg / mL) or DC (5 μg / mL) was added to each well, wherein the concentration of LDH in each group was maintained at 1, 2.5, 5, 10, 25, 50, 100, 250 μg / mL, and the nanomedicine was co-cultured with the cells at 5% CO2 and 37°C for 24 h. After that, the plate was removed, the original medium was discarded, washed three times with PBS, and fresh medium containing 10% (v / v) CCK-8 was added, and the cells were incubated in the incubator for 3 h. Finally, the absorbance of each well was measured at a wavelength of 450 nm using a multifunctional microplate reader, and the cells treated with PBS were used as blank controls, and the cell viability was recorded as 100%. Fig.12As shown in (A), as the concentration of LDH increases, the cell viability gradually decreases, which may be due to the Fenton-like reaction of LDH to produce ·OH, thus achieving chemodynamic therapy. There was no significant difference in the cell viability between the LDH-DC-BSA group and the LDH group within a certain concentration range. The cells in the LDH-LOX group had lower cell viability than those in the LDH group and the LDH-DC-BSA group, indicating that the anti-tumor effect of LDH-LOX was enhanced. The reason may be that LOX converts lactate into pyruvate and H2O2, thereby enhancing the chemodynamic therapy effect of LDH. Compared with LDH-LOX, LDH-DC-LOX showed a stronger anti-tumor effect, which may be due to the fact that DC inhibited the efflux of lactate, thereby further increasing the amount of intracellular H2O2, thereby enhancing the chemodynamic therapy effect. Fig.12 (B) shows that DC and LOX have no obvious toxicity to 4T1 cells under the treatment conditions. After LDH was loaded with DC and LOX, its anticancer activity was significantly enhanced.

[0092] Example 12

[0093] 4T1 cells were used as a model to detect the lactate efflux inhibition and lactate degradation effects of LDH-DC-LOX prepared in Example 1, LDH and LDH-LOX prepared in Comparative Example 1, and LDH-DC-BSA prepared in Comparative Example 2. 4T1 cells in the logarithmic growth phase were collected and 2×10 cells were added to each well. 5 The cells were seeded at a density of 100 cells / mL in a 6-well plate. The culture medium used was DMEM complete culture medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin and 10% FBS. The cells were incubated at 5% CO2 and 37°C for 24 hours. The original culture medium was discarded, and culture medium containing DC (5 μg / mL), LOX (0.2 μg / mL), LDH, LDH-DC-BSA, LDH-LOX or LDH-DC-LOX (LDH concentration was 10 μg / mL) was added to each well plate and co-cultured with the cells at 5% CO2 and 37°C for 12 hours. The lactate content in the cells and the cell culture medium was detected using a lactate detection kit. The extracellular lactate content was as shown in Fig.13 As shown in (A), because DC can inhibit lactate efflux, the extracellular lactate content of the DC group, LDH-DC-BSA group and LDH-DC-LOX group was significantly reduced. Because LOX can degrade lactate, the extracellular lactate content of the LOX group, LDH-LOX group and LDH-DC-LOX group was also significantly reduced. Compared with the LDH-DC-LOX group, the LDH-LOX group had a lower extracellular lactate content, i.e., lactate content in the culture medium, due to the inhibitory effect of DC on lactate efflux. The results of intracellular lactate content detection are shown in Fig.13As shown in (B), the intracellular lactate content in the DC group increased significantly due to the inhibitory effect of DC on lactate efflux. The intracellular lactate content in the LOX group decreased. Compared with the LOX group, the intracellular lactate content in the LDH-LOX group decreased significantly, which may be because LOX can more easily enter the cell after being loaded on the surface of LDH, resulting in a lower intracellular lactate content. Compared with the LDH-LOX group, the intracellular lactate content in the LDH-DC-LOX group was slightly higher than that in the LDH-LOX group due to the inhibitory effect of DC on lactate efflux.

[0094] Embodiment 13

[0095] 4T1 cells were used as a model to detect the effects of LDH-DC-LOX prepared in Example 1, LDH and LDH-LOX prepared in Comparative Example 1, and LDH-DC-BSA prepared in Comparative Example 2 on intracellular GSH levels. 4T1 cells in the logarithmic growth phase were collected and 2×10 5 The cells were seeded at a density of 100 cells / mL in a 6-well plate. The culture medium used was DMEM complete culture medium supplemented with 100U / mL penicillin, 100U / mL streptomycin and 10% FBS. The cells were incubated at 5% CO2 and 37°C for 24 hours. The original culture medium was discarded, and culture medium containing DC (5μg / mL), LOX (0.2μg / mL), LDH, LDH-DC-BSA, LDH-LOX or LDH-DC-LOX (LDH concentration was 10μg / mL) was added to each well and co-cultured with the cells at 5% CO2 and 37°C for 12 hours. The lactic acid content of intracellular GSH was detected using a GSH detection kit. The results are shown in Fig.14 As shown in Figure 2, LDH can significantly reduce the content of intracellular GSH, which may be due to the Cu released by LDH. 2+ Reduced to Cu by GSH in cells + The further decrease of intracellular GSH in LDH-LOX and LDH-DC-LOX groups may be due to the degradation of lactate into H2O2 by LOX, which promotes the + Further Fenton-like reactions occur to produce ·OH, which in turn removes GSH.

[0096] Embodiment 14

[0097] 4T1 cells were used as a model to qualitatively evaluate the effects of different materials on intracellular reactive oxygen species (ROS) levels.

[0098] (1) Flow cytometry was used to detect the level of ROS in cells. 5The cells were inoculated into a 6-well plate at a density of 100 cells / mL. The culture medium used was DMEM complete culture medium supplemented with 100U / mL penicillin, 100U / mL streptomycin and 10% FBS. The plates were incubated at 5% CO2 and 37°C for 24 hours. The original culture medium was discarded, and culture medium containing DC (5μg / mL), LOX (0.2μg / mL), LDH, LDH-DC-BSA, LDH-LOX or LDH-DC-LOX (LDH concentration was 10μg / mL) was added to each well and co-cultured with the cells at 5% CO2 and 37°C for 6 hours. The original culture medium was discarded, and the cells were co-incubated with the DCFH-DA probe diluted with serum-free culture medium for 30 minutes. The colorless DCFH-DA probe can be oxidized by ROS into DCF with green fluorescence in the cells. After the incubation, the original culture medium was discarded, and the plates were washed three times with PBS. Then the cells in all wells were digested, centrifuged, collected, and resuspended in PBS. The intensity of green fluorescence in the cells was detected by flow cytometry. Fig.15 As shown in the figure, DC and LOX did not affect the content of intracellular ROS. The intracellular ROS levels in the LDH group and LDH-DC-BSA group were significantly enhanced, indicating that LDH can produce ROS by Fenton-like reaction in cells. Compared with LDH, the cellular ROS levels in the LDH-LOX and LDH-DC-LOX groups were further increased, and LDH-DC-LOX showed the highest ROS level, indicating that H2O2 produced by lactate degradation can increase the level of ROS production, while DC inhibited lactate efflux, providing LOX with more degradation substrate lactate, thereby enhancing the Fenton-like reaction of the material.

[0099] (2) The intracellular ROS level was detected using a laser confocal microscope. 5 The cells were inoculated into a confocal cell culture dish at a density of 100 cells / mL. The culture medium used was DMEM complete culture medium supplemented with 100U / mL penicillin, 100U / mL streptomycin and 10% FBS, and incubated at 5% CO2 and 37°C for 24h. The original culture medium was discarded, and a culture medium containing DC (5μg / mL), LOX (0.2μg / mL), LDH, LDH-DC-BSA, LDH-LOX or LDH-DC-LOX (LDH concentration was 10μg / mL) was added to each well and co-cultured with the cells at 5% CO2 and 37°C for 6h. The original culture medium was discarded, and the cells were co-cultured with the DCFH-DA probe diluted in serum-free culture medium for 30min. The culture medium was discarded, and after washing three times with PBS, the cells were fixed with 2.5% glutaraldehyde solution for 15min, washed three times with PBS, and stained with DAPI for 10min. After that, the cells were washed three times with PBS, and then observed by laser confocal microscopy. The DCFH-DA probe can be oxidized by ROS into DCF showing green fluorescence in the cell. The results are as follows Fig.16As shown in the figure, consistent with the results of flow cytometry, there was no significant change in the intracellular ROS levels in the DC and LOX groups. The intracellular fluorescence intensity of the LDH and LDH-DC-BSA groups was significantly enhanced, and the ROS level in the LDH-LOX group was further enhanced, indicating that the loaded lactate can increase H2O2 by degrading lactate, thereby enhancing the ROS level. After loading DC, the efflux of intracellular lactate can be reduced, thereby increasing the intracellular lactate content and achieving further lactate degradation, thereby generating H2O2 and enhancing the Fenton-like reaction. Therefore, the cells in the LDH-DC-LOX group showed the highest ROS level.

[0100] Embodiment 15

[0101] 4T1 cells were used as a model to evaluate the effects of different materials on the level of intracellular lipid peroxides (LPO). 5 The cells were inoculated into a confocal cell culture dish at a density of 100 cells / mL. The culture medium used was DMEM complete culture medium supplemented with 100U / mL penicillin, 100U / mL streptomycin and 10% FBS. The cells were incubated at 5% CO2 and 37°C for 24h. The original culture medium was discarded, and the culture medium containing DC (5μg / mL), LOX (0.2μg / mL), LDH, LDH-DC-BSA, LDH-LOX or LDH-DC-LOX (LDH concentration was 10μg / mL) was added to each well and co-cultured with the cells at 5% CO2 and 37°C for 6h. The original culture medium was discarded, and the cells were co-incubated with C11-BODIPY 581 / 591 probe diluted with serum-free culture medium for 20min. The culture medium was discarded, and the cells were washed three times with PBS, fixed with 2.5% glutaraldehyde solution for 15min, washed three times with PBS, stained with DAPI for 10min, washed three times with PBS, and then observed by laser confocal microscopy. When the probe shows red fluorescence, it means that the probe has not been oxidized by LPO; when the probe shows green fluorescence, it means that the probe has been oxidized by LPO produced in the cell. Fig.17 As shown in AB, the fluorescence images and quantitative analysis results are consistent with the ROS results. The DC and LOX groups showed obvious red fluorescence and very weak green fluorescence, indicating that the intracellular LPO level was not significantly enhanced. The red fluorescence of cells co-incubated with LDH and LDH-DC-BSA was significantly weakened, and the green fluorescence was significantly enhanced, indicating that the intracellular LPO level was significantly enhanced. Due to the presence of DC and LOX, the cellular LPO level of LDH-LOX and LDH-DC-LOX groups was further enhanced, and the LPO level of LDH-DC-LOX group was the highest.

[0102] Example 16

[0103] 4T1 cells were used as a model to evaluate the apoptosis-promoting effects of different materials. Cells in the logarithmic growth phase were collected and 1×10 5 The cells were seeded at a density of 100 cells in a 12-well plate and incubated at 5% CO2 and 37°C for 24 hours. The original culture medium was discarded, and culture medium containing DC (5 μg / mL), LOX (0.2 μg / mL), LDH, LDH-DC-BSA, LDH-LOX or LDH-DC-LOX (LDH concentration is 10 μg / mL) was added to each well and co-cultured with the cells at 5% CO2 and 37°C for 24 hours. After that, the original culture medium was discarded, the cells were washed three times with PBS, and then digested with trypsin. The cells were collected by centrifugation at 1000 rpm for 5 minutes, and the cells were resuspended with PBS. The cells were stained with an apoptosis detection kit under light-proof conditions. 5 μL of Annexin V-FITC stain was added to each tube, and the cells were incubated at room temperature for 10 minutes. Then, 10 μL of PI staining solution was added, and the cells were incubated at room temperature in the dark for 5 minutes. Finally, flow cytometry was used to detect cell apoptosis. Fig.18 As shown in the flow cytometry dot plot of (A) and the quantitative analysis of 18 (B), the amount of apoptosis in the PBS group, DC group, and LOX group was low, while the amount of apoptotic cells in the LDH group and LDH-DC-BSA group increased significantly. The number of apoptotic cells in the LDH-LOX group increased further, indicating that LOX can enhance the effect of chemodynamic therapy. The apoptosis rate in the LDH-DC-LOX group was the highest, indicating that the intracellular lactic acid content increased under the action of DC, and degraded to produce H2O2 under the action of LOX, thereby further enhancing the effect of chemodynamic therapy.

[0104] Embodiment 17

[0105] A mouse subcutaneous tumor model was constructed to verify the in vivo antitumor activity of different materials. All animal experiments were approved by the Science and Technology Ethics Committee of Donghua University and were conducted in strict accordance with the standards of the Animal Protection Association. The 6-week-old female BALB / C mice used in the experiment were purchased from Shanghai Slake Laboratory Animal Center. 6 4T1 cells were inoculated into the right hind leg of the mouse, and the tumor was inoculated when the volume reached about 100 mm. 3When the mice were about 2 , they were randomly divided into 7 groups (6 mice in each group). The injection conditions for each group were as follows: control group (PBS); DC (2.5 mg / kg); LOX (0.1 mg / kg); LDH, LDH-DC-BSA; LDH-LOX; LDH-DC-LOX. The components containing LDH were injected with the nano-drug at an LDH concentration of 5 mg / kg. The injection dose for each group was 0.1 mL, and the injection method was intratumoral injection. The day when the treatment started was recorded as day 1. The materials of each group were intratumorally injected for treatment on days 1, 3, 5, and 7. During the treatment, the body weight and tumor size of the mice were recorded every 2 days. The length of the mouse tumor was recorded as L, and the width was recorded as W. Then the tumor volume was (L × W 2 ) / 2. After the treatment was over, the tumor tissues and main organs of the mice were collected for individual and tissue-level analysis.

[0106] (1) As Fig.19 (A) It can be seen that the body weights of the mice in each group gradually increased, indicating that LDH-DC-LOX and other materials had good biocompatibility in vivo. The relative tumor volume of each group ( Fig.19 B) showed that the tumor volume of the mice in the PBS group increased rapidly with time. Compared with the PBS group, the tumor growth of the mice in the other groups was inhibited to varying degrees. The order of their tumor inhibition rates was LOX group (23.1%) < DC group (30.6%) < LDH group (45.6%) < LDH-DC-BSA group (56.6%) < LDH-LOX (64.8%) < LDH-DC-LOX (79.3%). The tumor growth of the DC group and the LOX group was inhibited to a certain extent. The reason may be that DC inhibited the efflux of lactic acid, thus reducing the lactic acid content in the TME, while LOX degraded the lactic acid in the TME to reduce the lactic acid content. The decrease in the lactic acid content in the TME activated the immune system, thus producing a killing effect on tumor cells. The LDH group also showed a good tumor inhibition effect, indicating that LDH had a good chemodynamic therapy effect. Compared with the LDH group, LDH-DC-BSA had a better anti-tumor effect, which was due to the dual effects of chemodynamics and the activated immune system. In addition to the dual effects of chemodynamic therapy and immunotherapy, the LOX group in LDH-LOX degraded lactic acid to increase the intracellular H2O2 content, further promoting the chemodynamic therapy effect. The highest tumor inhibition rate of LDH-DC-LOX may be due to the fact that DC and LOX jointly inhibited the extracellular lactic acid content, making the lactic acid content in the TME lower. At the same time, DC increased the intracellular lactic acid content, resulting in more H2O2 produced by LOX degrading lactic acid, thereby enhancing the chemodynamic therapy effect. Fig.19 (C) The results of the tumor mass and volume changes in each group were consistent, and the tumor mass of the LDH-DC-LOX group was the smallest. The tumor blocks of each group were detected using a lactic acid detection kit. The results were as Fig. 20As shown, the lactic acid content in the tumors of the DC group, LOX group, LDH-DC-BSA group, LDH-LOX group, and LDH-DC-LOX group was significantly reduced. Under the dual effects of DC and LOX, the lactic acid content in the tumor mass of the LDH-DC-LOX group was the lowest.

[0107] (2) Slice analysis of tumors treated with different materials was performed, and H&E and TUNEL analysis were used to evaluate the necrosis and apoptosis of cells in the tumor site. The results of H&E staining showed that ( Fig.21 ), DC group, LOX group, LDH group, LDH group, LDH-DC-BSA group, LDH-LOX group, and LDH-DC-LOX group all showed cell fragmentation and nuclear condensation, among which the LDH-DC-LOX group had the least intact cells and the most obvious necrosis. TUNEL results showed ( Fig.21 ), each treatment group had different degrees of apoptosis, and the apoptosis in the LDH-DC-LOX group was the most obvious, which was consistent with the inhibition of tumor volume. The H&E staining results of the mouse lungs showed ( Fig.21 ), the PBS group had a larger lung tumor metastasis area, while the DC group, LOX group, LDH group, and LDH-DC-BSA group had a smaller lung metastasis area than the PBS group. The LDH-DC-LOX group had the least metastasis, indicating that the nanomedicine had a good anti-metastasis effect after treating tumors. Quantitative analysis of apoptosis Fig. 22 As shown, the trend of apoptosis level in each group was PBS (4.3%) <LOX(26.6%)<DC(31.8%)<LDH(47.3%)<LDH-DC-BSA(57.5%)<LDH-LOX(65.2%)<LDH-DC-LOX(72.5%)。

[0108] (3) Immunological analysis of the tumors and spleens of mice after treatment. The tumor and spleen tissues of each group of mice were removed under sterile conditions, cut into pieces and ground, and filtered through a 400-mesh filter to obtain a cell suspension. Lymphocytes were separated using a lymphocyte separation kit, and T lymphocyte suspension was separated using a nylon wool column. The collected T lymphocytes were labeled with different fluorescent antibodies and then analyzed by flow cytometry for CD3 in tumor tissues and spleen tissues. + CD4 + T cells (Anti-CD3-PE / Anti-CD4-FTIC), CD3 + CD8 + T cells (Anti-CD3-PE / Anti-CD8-FTIC) were quantitatively analyzed. In addition to flow cytometry analysis, the infiltration of immune cells in tumors and spleens was evaluated by section staining. Fig.23 (A), (B), and (E) show that CD3+ CD4 + T cells and CD3 + CD8 + The increase in the number of T cells may be due to the decrease in lactic acid content in the tumor microenvironment, which leads to the activation of the immune system and the enhancement of immune cell infiltration. The immune cells in the LDH group also increased slightly, which may be due to the acid degradation property of LDH, which neutralizes the acidity of TME, or chemodynamic treatment causes a certain immunogenic death, which activates T cells by maturing dendritic cells, leading to an increase in immune cell infiltration. + CD4 + T cells and CD3 + CD8 + T cells further increased, indicating that reducing the lactic acid content in tumors can significantly increase the infiltration of immune cells in tumors and activate the monitoring and killing function of the immune system. Fig.23 (C), (D), and (F) show that the immune CD3 + CD4 + T cells and CD3 + CD8 + The trend of T cell changes was consistent with that in tumor tissues, with the largest increase in immune cells in the LDH-DC-LOX group. These results indicate that degradation of lactic acid in the TME can activate the immune system and increase immune cell infiltration. In addition, staining of tumor and spleen tissue sections also visually showed that CD4 + T cells, CD8 + T cells increased significantly, and M2 macrophages in tumor tissues decreased significantly, while M1 macrophages and NK cells increased significantly ( Figure 24-25 ).

[0109] (4) After the treatment, the serum of each group of mice was collected. The immune factors in the serum were detected using an ELISA kit. The results were as follows: Fig.26 As shown, the secretion levels of granzyme B and interferon gamma (IFN-γ) anti-tumor proinflammatory cytokine (TNF-α) in the serum of mice in the LDH-DC-LOX group were the highest among all groups, indicating that the anti-tumor immune response effect in mice was better.

[0110] (5) Western blot analysis and oxidative damage analysis were performed on tumor tissues after treatment, including the expression of interferon regulatory factor 1 (IRF1), solute carrier family 7 member 11 (SLC7A11), one of the subunits of the cystine / glutamate transporter system Xc-, and glutathione peroxidase 4 (GPX4). Fig. 27As shown in the figure, after the treatment of tumors with each group of materials, the expression of interferon response protein IRF1 increased due to the increase of IFN-γ, and the increase was the largest in the LDH-DC-LOX group. The SLC7A11 and GPX4 proteins decreased the most in the LDH-DC-LOX group. The changes in the expression of the above proteins indicate that the increase in IFN-γ content will affect the uptake of cystine and glutamate from the extracellular space by tumor cells, thereby reducing the synthesis of intracellular GSH and further downregulating GPX4 protein, thereby promoting oxidative damage to cells. The GSH content of tumor tissue was detected using a GSH detection kit, and the results are shown in the figure. Fig.28 As shown in Figure 2, each treatment group was able to reduce the GSH content in the tumor, and the GSH content in the tumor of the mice in the LDH-DC-LOX group was the lowest. Then, the ROS detection kit and LPO detection kit were used for staining analysis. The results are shown in Figure 2. Fig.29 As shown in (A), (B), and (C), the ROS and LPO contents in the tumors of each treatment group increased, with the LDH-DC-BSA and LDH-LOX groups increasing more, and the LDH-DC-LOX group increasing the most, indicating that IFN-γ secreted by the activated immune system can further enhance the effect of chemodynamic therapy.

[0111] Embodiment 18

[0112] A mouse subcutaneous tumor model was constructed to investigate the metabolism of LDH-DC-LOX in mice. Six-week-old female BALB / C mice were purchased from Shanghai Slake Experimental Animal Center. 6 4T1 cells were inoculated into the right hind leg of the mouse, and the tumor volume reached about 300 mm. 3 At about 14:00 p.m., the mice were randomly divided into 2 groups (6 in each group) and kept in special mouse metabolic cages. The injection conditions of each group were as follows: control group (PBS, 0.1 mL) and experimental group LDH-DC-LOX (LDH concentration was 5 mg / kg, 0.1 mL), and the injection method was intratumoral injection. The urine and feces produced by mice injected with nanomedicines at 0h, 5-6h, 11-12h, 23-24h, 35-36h, and 47-48h were collected. The main organs (heart, liver, spleen, lung, kidney) and tumor tissues of the mice were collected at 48h. The main organs, tumor tissues, urine, and feces were digested with aqua regia, and then the copper content in the urine and feces of the mice was detected by inductively coupled plasma atomic emission spectrometry (ICP). Fig.30 (A) shows that within 48 hours after intratumoral injection of LDH-DC-LOX, there was no significant change in the copper content in mouse urine. Fig.30 (B) shows that the copper content in the feces of mice showed a trend of first increasing and then decreasing. Fig.30(C) shows that copper is still retained in the tumor 48 hours after the injection of the nanodrug. These results indicate that LDH-DC-LOX can be metabolized out of the body through feces after exerting its therapeutic effect and can remain in the tumor site for more than 48 hours.

[0113] Embodiment 19

[0114] In order to evaluate the biosafety of LDH-DC-LOX nanomedicine, the main organs of nude mice in each group after treatment in Example 17 were taken for H&E staining. The results showed that ( Fig.31 ) There was no obvious damage to the heart, liver, spleen, and kidney of the mice in each group, indicating that LDH-MTX@CM M -Ce6 nanomedicine has good biosafety. In addition, healthy BALB / c mice were randomly divided into 4 groups, 6 mice in each group, and 100μL of PBS, DC (2.5mg / kg), LOX (0.1mg / kg), and LDH-DC-LOX (LDH concentration is 5mg / kg) were injected through the tail vein. After 7 days, 3 nude mice in each group were collected for blood routine test through the orbital vein. After the remaining 3 nude mice in each group collected whole blood through the orbital vein, the serum was collected by centrifugation for blood biochemical analysis. After the LDH-DC-LOX nanomedicine was injected through the tail vein, the blood routine test results are shown in Figure (32). The number of white blood cells (WBC), the number of red blood cells (RBC), the hemoglobin content (HGB), and the number of platelets (PLT) were all within the normal range. The results of blood biochemical tests are shown in Figure (33). Liver function indicators including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and renal function indicators including uric acid (UA) and creatinine (CREA) were also within the normal range, indicating that LDH-DC-LOX nanomedicine has good biocompatibility and good clinical application prospects.

[0115] Comparative Example 1

[0116] (1) Prepare a 15 mL aqueous solution containing 10% DMSO but without DC. Add the 10% DMSO aqueous solution to a 15 mL solution containing MgCl2·6H2O (0.06M), CuCl2·6H2O (0.03M), and AlCl3·6H2O (0.045M). Slowly add NaOH (1M) solution to adjust the pH of the mixed solution to 9.5±0.2, and stir in an oil bath at 50°C in the dark for 24 h under a nitrogen atmosphere. After stirring, collect the sample by centrifugation at 3800g for 10 min, wash to remove free salt ions, and freeze-dry the final product to obtain Cu doped 2+ LDH NPs. Carry out relevant characterization and subsequent experiments.

[0117] (2) The LDH NPs solution in step (1) was mixed with the LOX solution at a mass ratio of 1:0.02, stirred at 37° C. in the dark for 4 h, and centrifuged at 3800 g for 10 min to obtain an LDH-LOX nanocomposite.

[0118] Comparative Example 2

[0119] (1) The LDH-DC NPs solution obtained in step (1) of Example 1 was mixed with the BSA solution at a mass ratio of LDH:BSA = 1:0.02, stirred at 37° C. in the dark for 4 h, and centrifuged at 3800 g for 10 min to obtain an LDH-DC-BSA nanocomposite.

Claims

1. A layered double hydroxide LDH composite material, characterized in that: The composite material comprises LDH nanomaterial doped with copper ions, drugs and lactate oxidase; wherein the LDH nanomaterial doped with copper ions is used as a carrier to load the drugs and modify the lactate oxidase.

2. The layered double hydroxide LDH composite material according to claim 1, characterized in that: The medicine includes diclofenac DC; the LDH nanomaterial doped with copper ions is LDH nanoparticles doped with copper ions.

3. The layered double hydroxide LDH composite material according to claim 1, characterized in that: The mass ratio of the copper ion-doped LDH nanomaterial to lactate oxidase is 0.9-1.1:0.01-0.

03.

4. A method for preparing the layered double hydroxide LDH composite material according to any one of claims 1 to 3, comprising: (1) mixing the drug, magnesium salt, copper salt, aluminum salt and water, adjusting the pH value, stirring the reaction under oil bath conditions, centrifuging, washing and freeze-drying to obtain a drug-loaded copper ion-doped LDH nanomaterial; (2) LOX, drug-loaded copper ion-doped LDH nanomaterials, and a solvent are mixed, stirred for reaction, centrifuged, and washed to obtain a layered double hydroxide LDH composite material.

5. The preparation method according to claim 4, characterized in that: In the step (1), the molar ratio of the drug, magnesium salt, copper salt and aluminum salt is 0.035-0.055:0.050-0.070:0.02-0.04:0.035-0.055; The drug in step (1) includes diclofenac DC; the magnesium salt includes MgCl2·6H2O; the copper salt includes CuCl2·2H2O; and the aluminum salt includes AlCl3·6H2O.

6. The preparation method according to claim 4, characterized in that: The drug in step (1) is a drug solution; the solvent of the drug solution is an aqueous solution containing 8-10% dimethyl sulfoxide (DMSO); and the adjusted pH value is 9.0-10.

0.

7. The preparation method according to claim 4, characterized in that: The stirring reaction under oil bath conditions in step (1) is carried out at 40-60° C. in the oil bath, in the dark, under nitrogen protection, and the stirring reaction time is 24-48 hours.

8. The preparation method according to claim 4, characterized in that: The solvent in step (2) is water; the stirring reaction is carried out at room temperature for 2 to 6 hours.

9. Use of the layered double hydroxide (LDH) composite material according to any one of claims 1 to 3 in the preparation of tumor nanomedicines for metabolic regulation and chemokinetics-immunotherapy.

10. Use of the layered double hydroxide (LDH) composite material according to any one of claims 1 to 3 in preparing a drug for treating breast cancer.

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