A dual-drug self-enhanced layered double hydroxide nanosystem and application thereof

Abstract

The application discloses a double-drug self-enhanced layered double hydroxide nanosystem and application thereof. The iron-ruthenium layered double hydroxide in the nanosystem has double enzyme activity, can efficiently catalyze H2O2 to generate O2 and active oxygen in a tumor microenvironment, significantly relieves hypoxia while inducing oxidative stress, the MCT4 specific inhibitor VB124 in the system creates an acidic microenvironment by blocking lactic acid output, amplifies the catalytic efficiency of FeRu, and establishes a self-enhanced catalytic effect; and the co-loaded epigenetic regulator JQ1 down-regulates the expression of PD-L1 by inhibiting BET protein. The system amplifies the catalytic efficiency of FeRu, significantly induces ferroptosis, reverses the immunosuppressive microenvironment with the help of epigenetic regulation, induces the release of damage-associated molecular patterns, causes the infiltration of immune cells, and finally cooperatively triggers a strong anti-tumor immune response. The self-enhanced catalysis-epigenetic regulation synergistic strategy realized by the system provides an innovative solution for improving the effect of tumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of antitumor immunotherapy technology, and in particular relates to a self-reinforcing layered double hydroxide nanosystem loaded with BRD4 inhibitor and MCT4 inhibitor and its application in antitumor immunotherapy. Background Technology

[0002] The tumor microenvironment (TME) possesses typical physicochemical characteristics such as hypoxia, high hydrogen peroxide (H2O2) concentration, and acidity. These characteristics are not only key factors driving malignant tumor progression but also provide potential targets for developing targeted therapy strategies. In particular, the H2O2 concentration in the TME can reach 5–10 times that of normal tissues, creating favorable conditions for developing H2O2-activated catalytic therapies. However, traditional Fenton catalysts (such as Fe...) 2+ Under the acidic and alkaline conditions of the tumor microenvironment (TME), catalytic activity is limited, making it difficult to efficiently utilize H2O2. Simultaneously, the highly expressed antioxidant defense system of tumor cells can rapidly scavenge reactive oxygen species (ROS), significantly weakening the efficacy of catalytic therapy. Furthermore, the spatiotemporal heterogeneity of the TME requires catalytic systems to possess environmental responsiveness and feedback regulation capabilities, which traditional catalysts struggle to achieve dynamic optimization of the catalytic process. On the other hand, given that immunotherapy relies on continuous immune activation signals, an ideal catalytic system should also possess the ability to provide sustained immune stimulation. Against this backdrop, the development of self-enhancing nanocatalytic systems is particularly important; such systems hold the promise of significantly improving therapeutic response and providing new insights into overcoming tumor drug resistance.

[0003] Layered hydrogen hydroxide (LDH) nanomaterials are a class of structurally regular two-dimensional inorganic materials that show great promise in biomedical fields such as tumor diagnosis and treatment, antibacterial applications, and tissue engineering due to their pH-responsive characteristics, good biocompatibility, and flexible drug loading capacity. Ruthenium iron oxide (FeRu) bimetallic nanomaterials, on the other hand, exhibit superior performance in catalyzing the decomposition of H₂O₂ to produce oxygen and generate ROS due to their unique electronic structure and highly efficient enzyme-like catalytic activity. However, single-component FeRu nanoparticles are still insufficient to completely reverse the hypoxic state in the tumor microenvironment (TME), and the explosive generation of ROS may induce adaptive drug resistance in tumor cells.

[0004] Bromodiphenyl ether domain protein 4 (BRD4), belonging to the BET protein family, is an important epigenetic regulator that drives the transcriptional activation of multiple key genes, including oncogenes, by recognizing acetylated histones and recruiting transcriptional complexes. BRD4 is abnormally highly expressed in various malignant tumors and is closely associated with tumor proliferation, metastasis, and treatment resistance. In the tumor microenvironment (TME), BRD4 can also promote tumor immune escape by regulating the expression of immune-related genes such as PD-L1 and IL-6. Therefore, targeting BRD4 has become a promising anti-tumor strategy. JQ1, as a potent and selective BET inhibitor, competitively binds to the bromodomain of BRD4, interfering with its interaction with acetylated histones, thereby inhibiting the transcription of oncogenes. This dual mechanism of action can simultaneously downregulate the expression of oncogenes and PD-L1 and modulate immune cell function. However, the clinical application of JQ1 is limited by its short plasma half-life, low oral bioavailability, and insufficient target specificity. Summary of the Invention

[0005] Therefore, the present invention aims to provide a self-enhancing layered double hydroxide nanosystem loaded with BRD4 and MCT4 inhibitors and its application in anti-tumor immunotherapy. In this invention, the iron-ruthenium layered double hydroxide nanosystem possesses dual-enzyme activity. The loaded MCT4 inhibitor VB124 amplifies the FeRu catalytic efficiency, establishing a self-enhancing catalytic effect; the co-loaded epigenetic regulator JQ1 downregulates oncogene and PD-L1 expression. This system significantly induces ferroptosis by amplifying FeRu catalytic efficiency, while simultaneously reversing the immunosuppressive microenvironment through epigenetic regulation, inducing the release of damage-associated molecular patterns (DAMPs), causing immune cell infiltration, and ultimately synergistically triggering a powerful anti-tumor immune response. The "self-enhancing catalysis-epigenetic regulation" synergistic strategy achieved by this system provides an innovative solution for improving the efficacy of tumor immunotherapy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a self-reinforced layered double hydroxide nanosystem loaded with a BRD4 inhibitor and an MCT inhibitor, comprising the following steps: (1) Under the protection of inert gas, an alkaline solution was added to a deoxygenated aqueous solution containing ferrous salt and ruthenium salt, and after stirring evenly, a hydrothermal reaction was carried out to obtain ferric-ruthenium layered bimetallic hydroxide nanoparticles. (2) The iron-ruthenium layered bimetallic hydroxide nanoparticles obtained in step (1) are dispersed in an acidic buffer solution for etching. The obtained nanoparticles are dispersed in water, and an alcohol solution containing BRD4 inhibitor and MCT inhibitor is added dropwise. The mixture is stirred for 1 to 10 hours and then sonicated. The resulting precipitate is redispersed in water, TPGS is added, and the mixture is stirred for 0.5 to 5 hours to obtain the final product.

[0008] Based on the above technical solution, the inert gas mentioned in step (1) further includes nitrogen, argon, helium and neon.

[0009] Based on the above technical solution, further, the ferrous salt mentioned in step (1) includes ferrous sulfate, nitrate, chloride and acetate, and the ruthenium salt includes ruthenium sulfate, nitrate, chloride and acetate.

[0010] Based on the above technical solution, further, the molar ratio of ferrous salt and ruthenium salt in the deoxygenated aqueous solution containing ferrous salt and ruthenium salt in step (1) is 1:0.1~1, preferably 1:0.4~0.6.

[0011] Based on the above technical solution, further, the concentration of ferrous salt in the deoxygenated aqueous solution containing ferrous salt and ruthenium salt in step (1) is 0.1~2.0 mM, preferably 0.3~0.5 mM.

[0012] Based on the above technical solution, the alkaline solution in step (1) further includes sodium hydroxide, potassium hydroxide and ammonia; the concentration is 2~10 mM, preferably 5~7 mM.

[0013] Based on the above technical solution, further, the stirring in step (1) is specifically stirring at 200~500 rpm for 10~60 min at room temperature.

[0014] Based on the above technical solution, further, the hydrothermal reaction in step (1) is specifically a hydrothermal reaction at 100~150℃ for 5~48h.

[0015] Based on the above technical solution, further, the volume ratio of the deoxygenated aqueous solution containing ferrous salt and ruthenium salt to the added alkaline solution in step (1) is 1:2~10, preferably 1:3~5.

[0016] Based on the above technical solution, further, the acidic buffer solution mentioned in step (2) is a PBS buffer solution with a pH of 3 to 5; the concentration of the iron ruthenium layered bimetallic hydroxide nanoparticles is 0.1 to 5 mg / mL, and the etching time is 2 to 24 h.

[0017] Based on the above technical solution, further, the concentration of the nanoparticle suspension in step (2) is 0.1~5 mg / mL.

[0018] Based on the above technical solution, further, the mass ratio of the BRD4 inhibitor and MCT inhibitor to the iron-ruthenium layered bimetallic hydroxide nanoparticles in step (2) is 0.5~2:0.5~2:1, preferably 1:1:1; the concentration of BRD4 inhibitor and MCT inhibitor in the alcohol solution containing BRD4 inhibitor and MCT inhibitor is 1~20 mg / mL.

[0019] Based on the above technical solution, further, the MCT4 inhibitor in step (2) is VB124 and the BRD4 inhibitor is JQ1. The iron-ruthenium nanoparticles loaded with VB124 can effectively achieve self-enhancement, and the dual drug loading can effectively promote anti-tumor immunotherapy.

[0020] Based on the above technical solution, further, the frequency of the ultrasound in step (2) is 20~30 kHz and the ultrasound time is 5~20 min.

[0021] Based on the above technical solution, further, the mass ratio of TPGS to precipitate in step (2) is 0.1~2:1; the concentration of precipitate in the dispersion is 0.1~5mg / mL.

[0022] Secondly, the present invention provides a self-reinforced layered double hydroxide nanosystem loaded with BRD4 inhibitor and MCT inhibitor prepared by the above preparation method.

[0023] Thirdly, the present invention provides the application of the above-mentioned self-reinforced layered double hydroxide nanosystem loaded with BRD4 inhibitor and MCT inhibitor in the preparation of drugs for treating tumors.

[0024] Based on the above technical solution, the tumor further includes melanoma.

[0025] The principle behind the application of the self-enhanced layered double hydroxide nanosystem loaded with BRD4 inhibitor and MCT inhibitor described in this invention in promoting anti-tumor immunotherapy is as follows: the iron-ruthenium layered double hydroxide nanoparticles have dual enzyme activity, the addition of VB124 effectively amplifies the FeRu catalytic efficiency, and the co-loading of JQ1 promotes the effect of immunotherapy.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention first prepares iron-ruthenium layered bimetallic hydroxide nanoparticles, then co-loads these nanoparticles with the BRD4 inhibitor JQ1 and the MCT4 inhibitor VB124. The iron-ruthenium layered bimetallic hydroxide exhibits dual-enzyme activity; FeRu efficiently catalyzes the decomposition of H2O2 into O2 and ROS in the TME, significantly alleviating hypoxia while inducing oxidative stress. VB124, as a specific inhibitor of MCT4, creates an acidic microenvironment by blocking lactate export and inducing significant intracellular acidification, amplifying the catalytic efficiency of FeRu and establishing a self-reinforcing cycle. The co-loaded epigenetic regulator JQ1 downregulates PD-L1 expression through BET protein inhibition. In vitro experiments confirm the simultaneous activation of multiple anti-tumor mechanisms: VB124-enhanced Fenton reaction generates a large amount of ROS, inducing ferroptosis, while in vivo experiments show that JQ1-mediated epigenetic reprogramming significantly increases CD8+. + By infiltrating T cells, reducing the proportion of Treg cells, and promoting dendritic cell maturation, this combined strategy achieved a tumor inhibition rate of 81.46% in a melanoma model, demonstrating excellent application prospects. Attached Figure Description

[0027] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0028] Figure 1 This is a transmission electron microscope (TEM) image of the FeRu nanoparticles from Example 1.

[0029] Figure 2 The graphs show the UV absorption curves and drug release curves of the JV@FeRu drug-loaded nanosystem in Example 1, where a is the UV absorption curve; b is the drug release curve of JQ1 at different pH values; and c is the drug release curve of VB124 at different pH values.

[0030] Figure 3 The graph shows the CAT activity detection results of FeRu nanoparticles in Example 2, where a is the evaluation of oxygen generation under different concentrations of H2O2, b is the quantitative measurement result of generated oxygen, c is the evaluation of oxygen generation under different concentrations of FeRu, and d is the quantitative measurement result of generated oxygen.

[0031] Figure 4 The figure shows the POD activity detection results of FeRu nanoparticles in Example 2. In the figure, a is the UV absorption curve of MB under different pH conditions; b is the UV absorption curve of MB in PBS at pH 6.5 at different times; c is the degradation of MB in PBS at pH 6.5 with different concentrations of FeRu nanoparticles.

[0032] Figure 5The graph shows the enzyme kinetics detection results of FeRu in Example 2, where ab are Michaelis-Menten and Lineweaver-Burk graphs of CAT enzyme activity, respectively; and cd are Michaelis-Menten and Lineweaver-Burk graphs of POD enzyme activity, respectively.

[0033] Figure 6 The graph shows the in vitro cytotoxicity and cell uptake results in Example 3, where a represents in vitro cytotoxicity and b represents cell uptake at different times.

[0034] Figure 7 The figure shows the results of in vitro cell enzyme activity verification in Example 3. In the figure, ab represents the quantitative analysis of O2 generation and average fluorescence intensity under different groups, respectively; cd represents the quantitative analysis of ROS generation and average fluorescence intensity under different groups, respectively.

[0035] Figure 8 The image shows the results of the in vitro antitumor immunoreaction analysis in Example 3. In the image, ab represents the immunofluorescence image and quantitative result of HMGB1, cd represents the immunofluorescence image and quantitative result of CRT, and ef represents the immunofluorescence image and quantitative result of PD-L1.

[0036] Figure 9 The figures shown are from Example 4, illustrating the establishment of the in vivo tumor model and the results of mouse body weight and mouse tumor volume. In the figures, a represents the establishment scheme of the mouse tumor model; b represents the changes in tumor volume of mice in each group; and c represents the changes in body weight of mice in each group.

[0037] Figure 10 The figure shows the results of the in vivo anti-tumor effect analysis. Figure a shows the immunohistochemical staining of tumor tissue with HE, Ki67, and PD-L1, and figure b shows the CD4+ staining. + T cells and CD8 + T-cell immunofluorescence staining image.

[0038] Figure 11 This is a flow cytometry analysis result of immune cells, where a represents mature dendritic cells (DCs) and b represents CD4+ cells. + T cells, c is CD8 + T cells, d represents Treg cells. Detailed Implementation

[0039] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0040] The room temperature mentioned in the examples refers to 25~30℃, and the water used is deoxygenated deionized water.

[0041] Example 1 Preparation and characterization of dual-loaded self-reinforced layered double hydroxide nanosystems 1. Iron-ruthenium layered bimetallic hydroxide nanoparticles A 10 mL deoxygenated deionized water solution containing 0.4 mM ferrous chloride and 0.2 mM ruthenium trichloride was prepared. N2 was bubbled through the solution, and 40 mL of a 6 mM sodium hydroxide aqueous solution was slowly added under stirring. The mixture was stirred at 300 rpm for 30 min at room temperature. Under N2 protection, the solution was added to a hydrothermal synthesis reactor and reacted at 120 °C for 24 h. The reaction product was a solution of iron-ruthenium layered bimetallic hydroxide nanoparticles (FeRu). The solution was centrifuged at 12000 rpm for 6 min, and the mixture was collected. The nanoparticles were washed three times with deionized water at the same speed. Finally, the nanoparticles were freeze-dried to constant weight to obtain layered bimetallic hydroxide nanoparticles, which were then analyzed by transmission electron microscopy (TEM). The results are shown in the figure below. Figure 1 As shown, the average particle size is 200 nm.

[0042] 2. Dually Loaded Self-Reinforced Layered Double Hydroxide Nanosystem The layered double hydroxide nanoparticles (FeRu) prepared in step 1 were dispersed in 2 mL of PBS at pH 4 at a concentration of 1 mg / mL. After etching at room temperature for 6 h, the mixture was centrifuged to remove the supernatant. The precipitate was redispersed in 2 mL of deionized water. Then, an ethanol solution containing VB124 and JQ1 (both VB124 and JQ1 were at a concentration of 8 mg / mL, with an added volume of 250 μL) was slowly added dropwise to the above suspension. The mass ratio of the carrier to VB124 was 1:1, and the mass ratio of the carrier to JQ1 was also 1:1. The mixture was magnetically stirred for 2 h, sonicated at 25 kHz for 10 min, and centrifuged at 12000 rpm for 6 min. The supernatant was removed, and the resulting precipitate was redispersed in 2 mL of water. 20 μL of TPGS (50 mg / mL) was added, and the mixture was magnetically stirred for 1 h to obtain the JV@FeRu suspension, which is the dual-loaded self-reinforced layered double hydroxide nanosystem. The same preparation method was used to prepare a single-loaded VB124 iron-ruthenium nano-suspension, namely VB124@FeRu, and the same preparation method was used to prepare a single-loaded JQ1@FeRu iron-ruthenium nano-suspension, namely JQ1@FeRu.

[0043] 3. Drug loading and release validation The drug complexes JV@FeRu suspension and VB124@FeRu suspension prepared in step 2 were diluted, and the iron-ruthenium layered bimetallic hydroxide nanoparticle suspension (FeRu) prepared in step 1 was used as a control. The absorbance of VB124, JQ1, VB124@FeRu and JV@FeRu was detected by ultraviolet absorption spectroscopy. The results are as follows. Figure 2 As shown in Figure a, compared with FeRu, VB124@FeRu has a similar absorption curve to VB124, proving that VB124 loading was successful. JV@FeRu has a similar maximum absorption peak to JQ1, proving that drug loading was successful.

[0044] 1 mL of JV@FeRu, VB124@FeRu, and JQ1@FeRu (1 mg / mL) suspensions were added to 2 mL of PBS buffer at different pH values. The solutions were then sealed in dialysis bags (Mw = 3500 Da) and immersed in 10 mL of PBS at pH 5.0, 6.5, and 7.4, respectively, with constant temperature shaking at 37°C. At different time points (0, 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h), 3 mL of PBS solution was removed from the dialysis bag, and fresh PBS buffer of the same pH was added simultaneously. The absorbance of the removed PBS solution was measured using a UV-Vis spectrophotometer at 254 nm and 238 nm. The concentrations of the drugs in the release media were calculated by substituting these values ​​into the JQ1 and VB124 standard curves. The results are as follows: Figure 2 As shown in bc, under acidic conditions (pH 5.0), JV@FeRu exhibited significant pH-responsive release behavior, with a 48-hour cumulative release rate of 93.81% for JQ1 and 89.91% for VB124, which were significantly higher than the release rates observed under neutral conditions (pH 7.4).

[0045] Example 2 Activity of iron-ruthenium layered bimetallic hydroxide nanoparticles determined 1.CAT activity The FeRu layered bimetallic hydroxide nanoparticles prepared in step 1 of Example 1 were dispersed in PBS solution at pH 6.5. The FeRu nanoparticles (100 μg / mL) were mixed with different concentrations of H2O2 (0, 5, 10, 40, 100 mM) and [Ru(dpp)3]Cl2 (5 μM). The mixture was placed in five replicates and incubated in a 96-well cell culture plate at 37°C for 20 min. After incubation, fluorescence intensity was recorded using an in vivo small animal imaging system (Ex=470 nm, Em=600 nm), and the fluorescence intensity of the oxygen probe at 620 nm was quantitatively detected using a multimode microplate reader. Different concentrations of FeRu nanoparticles (0, 25, 50, 100, 150, 300 μg / ml) were mixed with H2O2 (100 mM) and [Ru(dpp)3]Cl2 (5 μM) and incubated at 37 °C in 96-well cell culture plates for 20 min. After incubation, the results were detected using the same method described above. The results are as follows: Figure 3 As shown in the figure, the results indicate that with increasing H2O2 concentration, the FeRu nanoparticle concentration increases, and the CAT activity is significantly enhanced.

[0046] Enzyme kinetics: FeRu nanoparticles (100 μg / ml) were used as a catalyst in the presence of different concentrations of H2O2 (0, 10, 20, 30, 40, 50 mM). Dissolved oxygen concentration was recorded every 10 seconds using a dissolved oxygen meter.

[0047] For each H2O2 concentration, a scatter plot was created with H2O2 concentration on the x-axis and v on the y-axis, and then fitted using a Michaelis-Menten curve (Equation 2). Additionally, a linear reciprocal plot (Lineweaver-Burk plot, Equation 3) was used to determine... K m and V max ([S] is the substrate concentration) The results are as follows: Figure 5 As shown in ab, the CAT-like activity of FeRu nanoparticles follows the Michaelis-Menten model, with the Michaelis constant ( K m =76.329 mM) indicates moderate substrate affinity, but the maximum reaction rate ( V max =0.304 mg·L -1 ·s -1 It is superior to some natural enzymes, highlighting its effective catalytic performance.

[0048] 2. POD activity The FeRu nanoparticles prepared in step 1 of Example 1 were dispersed in a PBS solution at pH 6.5 to a concentration of 250 μg / mL. The formation of •OH from the FeRu nanoparticles was detected by Fenton reaction using methylene blue (MB) as an indicator. 4 mL of reaction solution was prepared. The FeRu nanoparticle suspension was mixed with H2O2 (100 mM) and MB (25 μg / mL) and dispersed in PBS at different pH values ​​(pH 4, 6.5, and 7.4). The UV-Vis-NIR absorption curves (400-800 nm) of the dispersed mixture were measured at different time points (0 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 12 h). To investigate the effect of FeRu nanoparticle concentration, FeRu nanoparticles (sample concentrations of 0, 50, 100, 250, and 500 μg / mL) were mixed with H2O2 (100 mM) and MB (25 μg / mL) in PBS (pH=4), and the absorption spectrum was measured after 4 hours of reaction. The results are as follows. Figure 4 As shown, Figure 4 a indicates that the lower the pH, the higher the POD activity. Figure 4 b is the UV absorption curve of MB at different times in PBS at pH 6.5; Figure 4 c shows that at pH 6.5, MB absorption gradually decreases with increasing FeRu nanoparticle concentration, indicating a significant enhancement of POD activity.

[0049] Enzyme kinetics: FeRu nanoparticles (250 μg / ml) were used as a catalyst in PBS (pH=6.5) containing MB (25 μg / ml) and different concentrations of H2O2 (0, 25, 50, 100, 200, 300 mM). The absorbance of MB was measured at 664 nm at different time points of 0, 10 min, 20 min, 30 min, 1 h, 2 h, 3 h, and 4 h.

[0050] For each H2O2 concentration, according to Beer-Lambert's law (Equation 1) (ε = 8.74 × 10⁻⁶), 4 The initial reaction rate (v0) was calculated based on the change in absorbance (L / (mol·cm)). The relationship between these rates and hydrogen peroxide content was plotted and then fitted using Michaelis-Menten curves (Equation 2). Furthermore, a linear reciprocal plot (Lineweaver-Burk plot, Equation 3) was used to determine... K m and V max ([S] is the substrate concentration) The results are as follows: Figure 5As shown in cd, the POD-like activity of FeRu nanoparticles conforms to Michaelis-Menten kinetics ( K m =112.208 mM, V max =1.553 10⁻⁷ M·s -1 Although its substrate affinity and catalytic rate are not as good as some natural enzymes, its extended catalytic stability gives it excellent ability to continuously generate ·OH.

[0051]

[0052]

[0053]

[0054] Example 3 In vitro antitumor effect analysis 1. Cytotoxicity test The cytotoxicity of the drug to cells was determined using a kit (CCK-8). B16 cells were cultured at 6 × 10⁶ cells / year. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates, with three replicates per well. The cells were incubated at 37 ºC for 24 hours in a 5% CO2 incubator. The culture medium was then changed, and different drug groups were added to each well: blank group (containing only culture medium for subsequent calculations), control group (containing only cells and culture medium), FeRu group, VB124@FeRu group, JV@FeRu group, and control group. The concentration gradients for the three experimental groups were 20, 40, 60, 80, and 100 μg / mL, respectively, and the cells were incubated for 12 hours. 10 μL of CCK-8 enhancement solution was added to each well, and after incubation for 1 hour, the absorbance was measured at 450 nm, calculated as (experimental group - blank group) / (control group - blank group). 100%, calculate cell viability. Cell viability results are as follows: Figure 6 As shown in figure a, the results show that JV@FeRu can effectively kill tumor cells.

[0055] 2. Cellular uptake The uptake of JV@FeRu by cells was observed by using FITC as a fluorescent probe instead of the drug in step 2 of Example 1 to study the uptake of the nanosystem. A density of 1×10⁻⁶ was used. 5B16 cells per mL were seeded into 12-well plates and incubated at 37°C for 24 hours. FITC-labeled drug loading agents at a concentration of 80 μg / mL were added and incubated for 1, 2, and 4 hours. Cells were then washed twice with PBS and labeled red with Actin Tracker 555 (200 μL). Subsequently, cell nuclei were stained with Hoechst 33342 (1 μg / mL) for 30 minutes, followed by washing twice with PBS. Finally, fluorescence images were captured using a CLSM. Results are shown below. Figure 6 As shown in b, the results indicate that JV@FeRu can be effectively taken up by cells.

[0056] 3. In vitro O2 generation assessment To assess intracellular oxygen production, B16 cells were subjected to a 1×10⁻⁶ thiocyanate inoculum. 5 Cells were seeded at a density of 1 cells / mL in 12-well plates containing cell slides and incubated for 24 hours. [Ru(dpp)3]Cl2 (7 μM) was added directly and incubated for 1 hour. The old culture medium was aspirated, washed with PBS, and 1 mL of new culture medium was added. Different test drugs were added: PBS (control group), 50 μg / mL FeRu, 50 μg / mL VB124@FeRu, and 50 μg / mL JV@FeRu. Cells were incubated for 4 hours, washed with PBS, fixed with paraformaldehyde for 15 minutes, and the nuclei were stained with Hoechst 33342 (1 μg / mL) for 30 minutes. Cells were washed twice with PBS, the slides were sealed, and finally, fluorescence images were captured using CLSM. Results are as follows: Figure 7 As shown in ab, the addition of VB124 effectively promotes the CAT enzyme activity of FeRu, and the red fluorescence is significantly quenched, indicating that more oxygen is produced.

[0057] 4. In vitro ROS generation assessment To assess intracellular ROS production, B16 cells were seeded into 12-well plates containing cell slides. After 24 hours of adherent growth, different drug groups were added: PBS (control group), 50 μg / mL FeRu, 50 μg / mL VB124@FeRu, and 50 μg / mL JV@FeRu. After 12 hours of drug incubation, the old medium was discarded, and 0.5 mL of DCFH-DA (10 μM) fluorescent probe was added to each well. The cells were incubated in the dark at 37°C for 30 minutes, washed twice with serum-free medium, fixed with 4% paraformaldehyde for 15 minutes, washed twice with serum-free medium, and then stained with Hoechst 33342 (1 μg / mL) to stain the nuclei. After incubation in the dark at 37°C for 30 minutes, fluorescence images were captured using CLSM. Results are as follows: Figure 7As shown in cd, the addition of VB124 effectively promotes the POD enzyme activity of FeRu, and the green fluorescence is significantly enhanced, indicating that VB124 effectively enhances the catalytic production of more ROS by FeRu, thus achieving self-enhanced catalysis of FeRu.

[0058] 5. Immune effect analysis To evaluate the effect of JV@FeRu on the immune response, B16 cells were seeded into 12-well plates containing cell slides and allowed to adhere for 24 hours. Different test drugs were then added: control PBS, FeRu (50 μg / mL), VB124@FeRu (50 μg / mL), and JV@FeRu (50 μg / mL). After 20 hours of incubation, cells were incubated with HMGB1, CRT, PD-L1 rabbit polyclonal antibody, and AF555-labeled donkey anti-rabbit IgG, respectively, followed by DAPI staining for 5 minutes. Finally, fluorescence images were captured using a CLSM scanner. Results are shown below. Figure 8 As shown in af, JV@FeRu can effectively downregulate PD-L1 expression, promote the recruitment of tumor-infiltrating T cells, and enhance the anti-tumor immune response. Furthermore, the release of HMGB1 and exposure to CRT indicate that JV@FeRu can effectively induce immunogenic cell death (ICD), thereby activating the systemic anti-tumor immune response.

[0059] Example 4 In vivo anti-tumor effect analysis 1. Construction of mouse tumor model All animal experiments were approved by the Animal Research Committee of Shenyang Pharmaceutical University. C57BL / 6 mice were purchased from Changsheng Biotechnology Co., Ltd. (Benxi, China). A xenograft B16 tumor model was established in female C57BL / 6 mice by subcutaneous injection of B16 cells.

[0060] 2. Experimental grouping and drug administration When the tumor volume reaches approximately 100 mm 3 At that time, the B16 tumor-bearing mice from step 1 were randomly divided into 4 groups: control group, FeRu group, VB124@FeRu group, and JV@FeRu group. On days 0, 3, 6, 9, and 12, each group of mice was injected intratumorally with saline (control group), FeRu (10 mg / kg), VB124@FeRu (10 mg / kg), and JV@FeRu (10 mg / kg), with an injection volume of 100 μL for each group. The treatment regimen was as follows. Figure 9 As shown in Figure a. Body weight and tumor volume were measured on days 0, 3, 6, 9, and 12. Mice were sacrificed on day 15. Changes in mouse body weight and tumor volume are shown in Figure a. Figure 9 As shown in bc. There was no significant difference in mouse body weight, and the JV@FeRu group effectively inhibited tumor volume, with a tumor inhibition rate as high as 81.46%.

[0061] 3. In vivo anti-tumor effect analysis and immunological analysis After treatment, tumors from each group were collected from euthanized mice for H&E, Ki67, and PD-L1 immunohistochemical staining, CD4 and CD8 immunofluorescence analysis, and flow cytometry was used to precisely quantify the proportion of key immune cells in the tumor immune microenvironment.

[0062] The results are as follows Figure 10-11 As shown, the tumor tissue in the JV@FeRu group exhibited typical necrosis characteristics such as large-area loose structure, nuclear pyknosis, and cell lysis in H&E staining. Figure 10 a). Ki67 immunohistochemistry showed a reduction in brown color in the JV@FeRu group, indicating a decrease in the proportion of proliferating cells, confirming its potent ability to inhibit tumor regeneration. PD-L1 immunohistochemistry results showed that JV@FeRu treatment reduced the brown color, indicating a significant decrease in PD-L1 expression levels on tumor cell membranes, suggesting that it may regulate and reverse the immunosuppressive microenvironment through epigenetic or signaling pathways. CD4 / CD8 immunofluorescence showed ( Figure 10 b) CD8 in the JV@FeRu group + T and CD4 + T cells were significantly increased in the JV@FeRu group compared to the control group, with higher CD8 counts. + T and CD4 + T cells showed significant co-localization in the tumor core region. Flow cytometry data ( Figure 11 Further clarification is needed regarding the JV@FeRu group's CD8... + The proportion of T cells rose to 31.8%, CD4 + The proportion of T cells rose to 32.3%, and Treg (CD4+) + / FOXP3 + The proportion decreased to 29.8%, accompanied by CD80. + / CD86 + The proportion of mature dendritic cells was significantly increased. These data collectively indicate that JV@FeRu can remodel the tumor immune microenvironment, promote effector T cell infiltration and activation, while inhibiting regulatory T cells and inducing a systemic anti-tumor immune response.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a self-reinforced layered double hydroxide nanosystem loaded with BRD4 inhibitor and MCT inhibitor, characterized in that, Includes the following steps: (1) Under the protection of inert gas, an alkaline solution was added to a deoxygenated aqueous solution containing ferrous salt and ruthenium salt, and after stirring evenly, a hydrothermal reaction was carried out to obtain ferric-ruthenium layered bimetallic hydroxide nanoparticles. (2) The iron-ruthenium layered bimetallic hydroxide nanoparticles obtained in step (1) are dispersed in an acidic buffer solution for etching. The obtained nanoparticles are dispersed in water, and an alcohol solution containing BRD4 inhibitor and MCT inhibitor is added dropwise. The mixture is stirred for 1 to 10 hours and then sonicated. The resulting precipitate is redispersed in water, TPGS is added, and the mixture is stirred for 0.5 to 5 hours to obtain the final product.

2. The preparation method according to claim 1, characterized in that, The inert gas mentioned in step (1) includes nitrogen, argon, helium and neon; the ferrous salt includes ferrous sulfate, nitrate, chloride and acetate; and the ruthenium salt includes ruthenium sulfate, nitrate, chloride and acetate.

3. The preparation method according to claim 1, characterized in that, The molar ratio of ferrous salt to ruthenium salt in the deoxygenated aqueous solution containing ferrous salt and ruthenium salt in step (1) is 1:0.1~1, preferably 1:0.4~0.6; the concentration of ferrous salt in the deoxygenated aqueous solution containing ferrous salt and ruthenium salt is 0.1~2.0 mM, preferably 0.3~0.5 mM.

4. The preparation method according to claim 1, characterized in that, The alkaline solution in step (1) includes sodium hydroxide, potassium hydroxide and ammonia; the concentration is 2~10 mM, preferably 5~7 mM.

5. The preparation method according to claim 1, characterized in that, The stirring in step (1) is specifically stirring at 200~500 rpm for 10~60 min at room temperature; the hydrothermal reaction is specifically a hydrothermal reaction at 100~150℃ for 5~48 h.

6. The preparation method according to claim 1, characterized in that, The acidic buffer solution mentioned in step (2) is a PBS buffer with a pH of 3 to 5; the concentration of the iron ruthenium layered bimetallic hydroxide nanoparticles is 0.1 to 5 mg / mL, and the etching time is 2 to 24 h.

7. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the nanoparticle suspension is 0.1~5 mg / mL; the mass ratio of the BRD4 inhibitor and MCT inhibitor to the iron-ruthenium layered bimetallic hydroxide nanoparticles is 0.5~2:0.5~2:1, preferably 1:1:1; the concentrations of the BRD4 inhibitor and MCT inhibitor in the alcohol solution containing the BRD4 inhibitor and MCT inhibitor are both 1~20 mg / mL.

8. The preparation method according to claim 1, characterized in that, In step (2), the MCT4 inhibitor is VB124 and the BRD4 inhibitor is JQ1; the frequency of ultrasound is 20~30 kHz and the ultrasound time is 5~20 min; the mass ratio of TPGS to precipitate is 0.1~2:1; and the concentration of precipitate in dispersion is 0.1~5 mg / mL.

9. A self-reinforced layered double hydroxide nanosystem loaded with BRD4 inhibitor and MCT inhibitor prepared by the preparation method according to any one of claims 1-8.

10. The use of the self-reinforced layered double hydroxide nanosystem loaded with BRD4 inhibitor and MCT inhibitor as described in claim 9 in the preparation of drugs for treating tumors.

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