A sugar metabolism regulation type nanomaterial, a preparation method thereof and application thereof in anti-tumor treatment

Abstract

The application discloses a sugar metabolism regulation type nanomaterial, a preparation method thereof and application thereof in anti-tumor treatment, and belongs to the technical field of tumor pharmaceutical preparations. The sugar metabolism regulation type nanomaterial provided by the application takes a double sulfur death / iron death inducer loaded with a chemotherapeutic drug as an inner core, and is surface-modified with a NO donor conjugate; the sugar metabolism regulator is a polyphenol compound, and the double sulfur death / iron death inducer is a metal-phenolic network, which can interfere with sugar metabolism, reduce NADPH production, thereby causing cystine accumulation and actin cytoskeleton destruction to induce double sulfur death; and inhibit the synthesis of GSH to enhance the iron death effect. In addition to the direct tumor killing effect, the nanomaterial can also reverse the immunosuppressive tumor microenvironment, realizing synergistic inhibition on tumor treatment. The application provides a new strategy with good anti-tumor effect and application potential for cancer combined treatment.

Description

Technical Field

[0001] This invention relates to the field of tumor drug formulation technology, specifically to a glucose metabolism regulating nanomaterial, its preparation method, and its application in anti-tumor therapy. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Tumor cells over-uptake nutrients such as glucose through metabolic reprogramming, leading to an increase in CD8+ in the tumor microenvironment. + The function of effector immune cells such as T cells is impaired. At the same time, the accumulation of metabolites such as lactate in the tumor microenvironment promotes the polarization of tumor-associated macrophages (TAMs) towards the M2 type, forming an immunosuppressive network and hindering the establishment of anti-tumor immunity.

[0004] The reprogramming of glucose metabolism in tumor cells also participates in regulating multiple cell death pathways. The pentose phosphate pathway-mediated generation of nicotinamide adenine dinucleotide phosphate (NADPH) is crucial for maintaining glutathione synthesis (GSH) and regeneration. In SLC7A11-overexpressing tumor cells, when glucose supply is sufficient, the active pentose phosphate pathway provides ample NADPH, supporting glutathione synthesis and regeneration, and enhancing cellular resistance to ferroptosis. When glucose supply is limited, insufficient NADPH generation leads to abnormal cysteine ​​accumulation and disulfide bond formation in the actin cytoskeleton, inducing disulfide death; it also weakens glutathione regeneration capacity, synergistically enhancing ferroptosis sensitivity. Further research is needed to determine how to provide effective anti-tumor strategies based on these metabolic mechanisms. Summary of the Invention

[0005] To address at least one of the technical problems existing in the background art, this invention provides a glucose metabolism-regulating nanomaterial, its preparation method, and its application in anti-tumor therapy. The nanomaterial provided by this invention exhibits excellent anti-tumor efficacy by regulating the glucose metabolism of tumor cells, synergistically enhancing disulfide death and ferroptosis effects, and effectively reversing the immunosuppressive tumor microenvironment.

[0006] The present invention adopts the following technical solution: In a first aspect, the present invention provides a glucose metabolism regulating nanomaterial, the glucose metabolism regulating nanomaterial comprising a metal-phenolic network core, a chemotherapeutic drug loaded in the metal-phenolic network core, and a nitric oxide donor conjugate modified on the surface of the metal-phenolic network core.

[0007] A second aspect of the present invention provides a method for preparing the above-mentioned sugar metabolism regulating nanomaterials, comprising the following steps: N-acetylcysteine ​​was added to an aqueous solution of hydrochloric acid containing sodium nitrite to react and obtain nitrosylated N-acetylcysteine. The nitrosylated N-acetylcysteine ​​was then activated by carboxylation and added dropwise to hyaluronic acid to prepare nitrosylated N-acetylcysteine-hyaluronic acid. Chemotherapy drug solution, metal salt solution and nitrosylated N-acetylcysteine-hyaluronic acid solution were stirred and mixed, and polyphenol compound solution was added to react. After dialysis and freeze-drying, sugar metabolism regulating nanomaterials were obtained.

[0008] A third aspect of the present invention provides the application of the above-described glucose metabolism regulating nanomaterials, or glucose metabolism regulating nanomaterials prepared by the above-described method, in the preparation of antitumor drugs, wherein the drugs comprise at least one or more of the following uses: 1) It inhibits glucose metabolism, blocks glucose uptake, disrupts the actin cytoskeleton, and induces disulfide death; 2) Consuming GSH downregulates GPX4 protein expression, inducing ferroptosis; 3) It produces NO, which promotes drug penetration and inhibits cell metastasis; 4) Reverse the immunosuppressive tumor microenvironment and improve anti-tumor efficacy.

[0009] A fourth aspect of the present invention provides an antitumor pharmaceutical composition comprising the above-mentioned glucose metabolism regulating nanomaterial and a pharmaceutically acceptable carrier; the dosage form of the pharmaceutical composition is at least one of an injection, a nanoparticle formulation, or a lyophilized powder injection.

[0010] Compared with the prior art, the beneficial effects of the present invention are: The glucose metabolism regulating nanomaterial provided by this invention uses a metal-phenolic network as its core, loads chemotherapeutic drugs within the core, and modifies the core surface with nitric oxide donor conjugates. This material effectively achieves a multi-mechanism synergistic antitumor effect by inhibiting tumor cell glucose metabolism, releasing NO, simultaneously inducing disulfide death and ferroptosis, and reversing the immunosuppressive tumor microenvironment, thus significantly inhibiting breast cancer growth. This invention provides a promising treatment strategy for the combination therapy of breast cancer, demonstrating excellent antitumor efficacy and clinical translational potential, and has promising application scenarios. Attached Figure Description

[0011] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0012] Figure 1 The particle size distributions of VE, VEP, VEP@HA-NO, VEP@HA-NO-2, VEP@HA-NO-3, and VEP@HA-NO-4 in this invention are shown below. Figure 2 This is a graph showing the stability results of VEP@HA-NO, VEP@HA-NO-2, VEP@HA-NO-3, and VEP@HA-NO-4 in PBS in Example 6 of the present invention. Figure 3 This is a graph showing the GSH residue results of VEP@HA-NO at different times in Example 7 of the present invention; Figure 4 This is a graph showing the NO release results after different treatments in Example 8 of the present invention; Figure 5 This is a graph showing the cytotoxicity results of VE, VEP, or VEP@HA-NO at different concentrations according to the present invention; Figure 6 This is a fluorescence image of ROS in 4T1 cells after different treatments in Example 10 of the present invention; Figure 7 This is a diagram showing the expression of GPX4 protein in 4T1 cells after different treatments in Example 11 of this invention; Figure 8 This is a graph showing the relative glucose content in the supernatant of 4T1 cells after different treatments in Example 12 of the present invention. Figure 9 This is a diagram showing the structural changes of actin in 4T1 cells after different treatments in Example 13 of the present invention. Detailed Implementation

[0013] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0014] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0015] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0016] In one typical embodiment of the present invention, a glucose metabolism regulating nanomaterial is provided, the glucose metabolism regulating nanomaterial comprising a metal-phenolic network core, a chemotherapeutic drug loaded in the metal-phenolic network core, and a nitric oxide donor conjugate modified on the surface of the metal-phenolic network core.

[0017] Furthermore, the nitric oxide donor conjugate is nitrosylated N-acetylcysteine-hyaluronic acid.

[0018] The metal-phenolic network core is formed by the coordination of metal salts and polyphenolic compounds. The coordination compound has pH-responsive properties and can be degraded due to the weakly acidic microenvironment of tumor cells and the acidic conditions in lysosomes, thereby releasing mixed valence vanadium and polyphenolic compounds, realizing intelligent response to the tumor microenvironment and controlled drug release.

[0019] Furthermore, the metal salt includes one of ammonium metavanadate, sodium orthovanadate, vanadium oxysulfate, or vanadium chloride; the polyphenolic compound includes one of tannic acid, gallic acid, or EGCG.

[0020] Among them, polyphenolic compounds are inhibitors of glucose metabolism, and metal-phenolic networks are inducers of disulfide death / ferroptosis.

[0021] Furthermore, the chemotherapy drug includes at least one of paclitaxel, docetaxel, doxorubicin, or cyclophosphamide.

[0022] This invention provides a novel therapeutic modality that synergistically enhances disulfide death and ferroptosis by modulating glucose metabolism in tumor cells. This strategy relies on the aforementioned glucose metabolism-regulating nanomaterials, which accumulate in tumor cells through CD44 receptor-mediated active targeting, releasing NO to promote drug penetration. Its mixed-valence vanadium (V... 4+ and V 5+This compound can trigger a Fenton-like reaction and consume GSH, thereby inducing ferroptosis in tumor cells. Simultaneously, polyphenolic compounds can regulate tumor cell glucose metabolism, effectively blocking glucose uptake and reducing NADPH production, thus producing a dual synergistic effect. On the one hand, it leads to cysteine ​​accumulation and actin cytoskeleton disruption, thereby inducing disulfide death; on the other hand, it also inhibits GSH synthesis and regeneration, thereby enhancing the ferroptosis effect. Furthermore, the glucose metabolism-regulating nanomaterials can also inhibit lactate efflux, reshape the immunosuppressive tumor microenvironment, reduce the proportion of M2 tumor-associated macrophages, and restore T lymphocyte activity. These multi-mechanism synergistic effects ultimately achieve significant inhibition of tumor growth.

[0023] This invention, for the first time, utilizes a metal-phenolic network to regulate glucose metabolism in tumor cells, achieving a simultaneous enhancement of disulfide death and ferroptosis, providing a novel strategy for the synergistic induction of disulfide death / ferroptosis at the metabolic intervention level. This approach can further activate anti-tumor immune responses, thereby enhancing the anti-tumor effect of the metabolically regulated nanosystem. This invention expands the application dimensions of glucose metabolism regulation in combination cancer therapy, possessing significant scientific value and clinical translational potential.

[0024] Another embodiment of the present invention provides a method for preparing the above-mentioned sugar metabolism regulating nanomaterial, comprising the following steps: N-acetylcysteine ​​was added to an aqueous solution of hydrochloric acid containing sodium nitrite to react and obtain nitrosylated N-acetylcysteine. The nitrosylated N-acetylcysteine ​​was then activated by carboxyl group and added dropwise to hyaluronic acid to prepare nitrosylated N-acetylcysteine-hyaluronic acid. Chemotherapy drug solution, metal salt solution and nitrosylated N-acetylcysteine-hyaluronic acid solution were stirred and mixed, and polyphenol compound solution was added to react. After dialysis and freeze-drying, sugar metabolism regulating nanomaterials were obtained.

[0025] Furthermore, the stirring reaction time is 6~36 h, more preferably 10~30 h; the stirring speed is 200~1000 rpm, more preferably 400~800 rpm; Furthermore, the dialysis specifically involves using a dialysis bag with a molecular weight cutoff of 1000~10000 Da for dialysis for 1~3 days. The present invention does not impose special restrictions on the dialysis process, as long as unreacted raw materials can be removed, thereby achieving the purpose of product purification.

[0026] Furthermore, freeze-drying is performed for 24-48 hours; the present invention does not impose any special restrictions on the freeze-drying method, and freeze-drying can be performed using a freeze dryer commonly used in the art.

[0027] Furthermore, the carboxyl activation specifically involves activating the carboxyl group of nitrosylated N-acetylcysteine ​​using dicyclohexylcarbodiimide and 4-dimethylaminopyridine.

[0028] Further, the molar ratio of the metal salt to the polyphenol compound is 1:(0.3~3), more preferably 1:(0.5~2); the molar ratio of the nitrosylated N-acetylcysteine ​​to hyaluronic acid is 1:(0.5~2).

[0029] Furthermore, the mass ratio of the metal salt, polyphenol compound, chemotherapeutic drug and nitrosylated N-acetylcysteine-hyaluronic acid is 1:(1~5):(0.5~2):(1~3).

[0030] A third embodiment of the present invention provides the application of the above-mentioned glucose metabolism regulating nanomaterial or the glucose metabolism regulating nanomaterial prepared by the above-mentioned method in the preparation of an antitumor drug, wherein the drug comprises at least one or more of the following uses: 1) It inhibits glucose metabolism, blocks glucose uptake, disrupts the actin cytoskeleton, and induces disulfide death; 2) Consuming GSH downregulates GPX4 protein expression, inducing ferroptosis; 3) It produces NO, which promotes drug penetration and inhibits cell metastasis; 4) Reverse the immunosuppressive tumor microenvironment and improve anti-tumor efficacy.

[0031] Furthermore, the cancer is breast cancer, and even more specifically, breast cancer with high expression of SLC7A11.

[0032] A fourth embodiment of the present invention provides an antitumor drug composition comprising the above-mentioned glucose metabolism regulating nanomaterial and a pharmaceutically acceptable carrier; the dosage form of the drug composition is at least one of an injection, a nanoparticle formulation, or a lyophilized powder injection.

[0033] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0034] In the following examples, the culture medium for the Control group was RPMI 1640 medium (Gibco, USA); the culture media containing PTX, VE, VEP, VEP@HA or VEP@HA-NO were used to add PTX, VE, VEP, VEP@HA or VEP@HA-NO to the Control group, and the drug concentration of VEP@HA-NO was controlled at 50 μg / mL.

[0035] Example 1 This embodiment provides a nanomaterial for regulating sugar metabolism.

[0036] 1.1 Preparation of metal-phenolic network (VE) EGCG (229.2 mg, 0.5 mmol) was dissolved in 10 mL of deionized water to obtain solution A. NH4VO3 (58.5 mg, 0.5 mmol) was dissolved in 10 mL of deionized water under heating in a 60 °C water bath to obtain solution B. 1 mL of solution A and solution B were added to flasks containing 8 mL of PBS (pH 7.4), and the mixtures were reacted at 700 rpm for 12 h. The resulting solutions were dialyzed against deionized water for 48 h (MW = 3.5 kDa) to remove unreacted reactants. The solutions were then freeze-dried to obtain vitamin E.

[0037] 1.2 Preparation of drug-loaded metal-phenolic networks (VEP) 50 mg of PTX was dissolved in 2.5 mL of DMSO to obtain solution C. 250 μL of solution C and 1 mL of solution B were added to a flask containing 8 mL of PBS (pH 7.4), and the mixture was stirred at 500 rpm for 20 min. Then, 1 mL of solution A was added, and the reaction was continued at 500 rpm for 12 h. The resulting solution was dialyzed against deionized water for 48 h (MW = 3.5 kDa) to remove unreacted raw materials. After freeze-drying, VEP was obtained.

[0038] 1.3 Preparation of NO donor conjugates NAC was added to an equimolar amount of sodium nitrite (NaNO2) in an aqueous HCl solution and reacted in an ice bath in the dark for 40 min. After adding acetone, washing with cold water, vacuum filtration, and freeze-drying, nitrosylated NAC was obtained. The nitrosylated NAC (0.30 mmol / L) was dissolved in 10 mL of DMF and reacted with dicyclohexylcarbodiimide (DCC) (0.30 mmol / L) and 4-dimethylaminopyridine (DMAP) (0.05 mmol / L) in an ice bath in the dark for 1 h to activate the carboxyl group of NAC. HA (95 mg, molecular weight 10 kDa, containing approximately 0.25 mmol / L of COOH) was dissolved in 10 mL of formamide, and NAC was added dropwise to the solution. The mixture was stirred at room temperature in the dark for 24 h under N2 protection. The resulting solution was dialyzed against distilled water in the dark (MW = 3.5 kDa) for 2 days. The solution was then filtered and freeze-dried to obtain HA-NO, which was stored at 4°C for later use.

[0039] 1.4 Preparation of Nanomaterials Regulating Glucose Metabolism Add 250 μL of solution C, 1 mL of solution B, and 10 mg of HA-NO to a flask containing 8 mL of PBS (pH 7.4). Stir at 500 rpm for 20 min, then add 1 mL of solution A and continue the reaction at 500 rpm for 12 h. Dialyze the resulting solution with deionized water for 48 h (MW = 3.5 kDa) to remove unreacted raw materials. Freeze-dry the product to obtain VEP@HA-NO.

[0040] Comparative Example 1 Preparation of VEP@HA: Add 250 μL of solution C, 1 mL of solution B, and 10 mg of HA to a flask containing 8 mL of PBS (pH 7.4). Stir at 500 rpm for 20 min, then add 1 mL of solution A and continue the reaction at 500 rpm for 12 h. Dialyze the resulting solution with deionized water for 48 h (MW = 3.5 kDa) to remove unreacted raw materials. Freeze-dry the product to obtain VEP@HA.

[0041] Example 2 The preparation method was the same as in Example 1, except that EGCG was replaced with an equimolar amount of gallic acid (85.1 mg, 0.5 mmol). All other steps were the same, yielding VEP@HA-NO-2.

[0042] Example 3 The preparation method was the same as in Example 1, except that ammonium metavanadate was replaced with vanadium oxysulfate (81.5 mg, 0.5 mmol). All other steps were the same, yielding VEP@HA-NO-3.

[0043] Example 4 The preparation method was the same as in Example 1, except that ammonium metavanadate was replaced with vanadium chloride (78.7 mg, 0.5 mmol). All other steps were the same, yielding VEP@HA-NO-4.

[0044] Example 5 Particle size detection of nanomaterials regulating glucose metabolism: The VE, VEP, VEP@HA-NO prepared in Example 1, VEP@HA-NO-2 prepared in Example 2, VEP@HA-NO-3 prepared in Example 3, and VEP@HA-NO-4 prepared in Example 4 were dispersed in 5 mL of PBS, and their hydrated particle size was measured. The results are as follows. Figure 1As shown, the average particle size of VE is 117.2 nm, the average particle size of VEP is 132.5 nm, the average particle size of VEP@HA-NO is 156.4 nm, the average particle size of VEP@HA-NO-2 is 321.1 nm, the average particle size of VEP@HA-NO-3 is 405.7 nm, and the average particle size of VEP@HA-NO-4 is 285.7 nm.

[0045] It is evident that, compared to VE, VEP exhibits a larger particle size due to the loading of PTX; further surface modification with HA-NO leads to an even greater increase in the particle size of VEP@HA-NO, a trend consistent with the material's structural modification process.

[0046] Example 6 Stability assessment of nanomaterials regulating glucose metabolism: VEP@HA-NO, VEP@HA-NO-2, VEP@HA-NO-3, and VEP@HA-NO-4 were each uniformly dispersed in 10 mL of PBS (pH=7.4) and stored at 4 ℃. The hydrated particle size of the glucose metabolism-regulating nanomaterials was measured at the same time point daily to assess their physical stability.

[0047] The results are as follows Figure 2 As shown, the particle size of the glucose metabolism regulating nanomaterial did not change significantly within 7 days, indicating that the formulation has good dispersion stability.

[0048] Example 7 Assess the GSH consumption of VEP@HA-NO in aqueous solution: The ability of VEP@HA-NO to consume GSH was evaluated using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). 1 mL of 100 μg / mL VEP@HA-NO solution prepared in Example 1 was mixed with an equal volume of 10 mM GSH and incubated for 0, 1, 2, 4, and 8 h, respectively. After centrifugation at 10000 rpm for 5 min, 1 mL of the supernatant was added to 5 mM DTNB solution, and the absorbance at 412 nm was measured.

[0049] The results are as follows Figure 3 As shown, the residual GSH content in the solution gradually decreased with the extension of co-incubation time between VEP@HA-NO and GSH. After 8 h of co-incubation, the residual GSH content was less than 50% of the initial value, indicating that VEP@HA-NO has excellent GSH consumption capacity, and this consumption effect is time-dependent.

[0050] Example 8 NO release capacity assessment: The NO release capacity of VEP@HA-NO was determined using a NO content detection kit. VEP@HA, VEP@HA+H2O2, VEP@HA-NO, and VEP@HA-NO+H2O2 were incubated at 37 ℃ for 0.5, 1, 1.5, or 2 h, respectively. 50 μL of the supernatant was taken, and equal volumes of Griess reagents I and II from the NO content detection kit were added sequentially. After reacting at room temperature in the dark for 10 min, the absorbance at 540 nm was measured using a microplate reader, and the NO release amount of each sample was calculated.

[0051] Experimental results are as follows Figure 4 As shown, no significant NO release was detected in VEP@HA regardless of the presence of H2O2 in the system, which is consistent with the fact that the material itself does not contain a NO donor structure. In contrast, VEP@HA-NO exhibits significant H2O2-responsive release behavior: in the absence of H2O2 stimulation, NO release is slow, which helps maintain the stability of the nanoparticles in bulk circulation and avoids premature NO leakage; while in the presence of H2O2, VEP@HA-NO can rapidly release a large amount of NO, showing obvious H2O2-triggered release characteristics.

[0052] Example 9 Cytotoxicity assessment: 4T1 cells were seeded at a density of 7000 cells per well in 96-well plates and cultured for 24 h. The original culture medium was discarded, and 100 μL of medium containing different concentrations (25, 50, 75, 100, 125 μg / mL) of VE, VEP, or VEP@HA-NO was added to each well, and incubation continued for another 24 h. After incubation, the drug-containing medium was discarded, and the cells were washed 2-3 times with PBS. 100 μL of medium containing 10% CCK8 was added to each well, and after incubation for 1 h, the absorbance was measured at 450 nm.

[0053] The results are as follows Figure 5 As shown, VE, VEP, or VEP@HA-NO all exhibited dose-dependent cytotoxicity against 4T1 cells. At a drug concentration of 75 μg / mL, single application reduced cell viability by 34.5%; while VEP showed a stronger inhibitory effect, reducing cell viability by 61.3%. VEP@HA-NO, containing a NO donor, further reduced cell viability at this concentration, achieving a tumor inhibition rate of 72.0%, indicating its excellent cytotoxicity.

[0054] Example 10 Intracellular ROS content measurement: 4T1 cells were fed at a rate of 2 × 10 5Cells were seeded at a density of / well in 6-well plates and cultured overnight. Cells were then incubated for 24 h in medium containing PTX, VE, VEP, VEP@HA, or VEP@HA-NO (50 μg / mL), respectively. The medium was discarded, and after washing with PBS, 1 mL of DCFH-DA (10 μM) fluorescent probe working solution was added, and the cells were incubated in the dark for 20 min. Cells were washed 2-3 times with PBS to remove excess probe, and then the intracellular ROS fluorescence intensity was observed using an inverted fluorescence microscope.

[0055] The results are as follows Figure 6 As shown, compared with the control group and the PTX group, cells treated with VE, VEP, and VEP@HA all exhibited significant green fluorescence signals, indicating a significant increase in intracellular ROS levels. This sharp increase in ROS mainly stemmed from the large amount of ·OH generated by the Fenton-like reaction of the nanoparticles, and the ROS accumulation caused by the dysregulation of the cellular antioxidant system after GSH depletion. Notably, the VEP@HA-NO group did not show the expected high level of ROS accumulation, possibly because the NO released from it reacted with ROS to convert into reactive nitrogen species (RNS), thereby reducing the net accumulation of ROS.

[0056] Example 11 Assessment of GPX4 protein expression: 4T1 cells were fed at a dose of 1×10 5 Cells were seeded at a density of / wells onto 12-well cell culture slides and cultured until adherent. Medium containing PTX, VE, VEP, VEP@HA, or VEP@HA-NO (50 μg / mL) was added to each well, and incubation continued for 24 h. After incubation, the drug-containing medium was discarded, and the cells were washed three times with PBS. The cells were then fixed sequentially with 4% paraformaldehyde for 15 min, blocked with 5% goat serum for 30 min, incubated with GPX4 monoclonal antibody for 1 h, incubated with AF488-labeled goat anti-rabbit IgG in the dark for 1 h, and stained with DAPI for 5 min. Intracellular GPX4 expression was then observed using a confocal microscope.

[0057] The results are as follows Figure 7 As shown, bright green fluorescence signals were observed in the control group and PTX group, while the intensity of green fluorescence in cells was significantly reduced after treatment with VE, VEP, VEP@HA and VEP@HA-NO, indicating that these agents can effectively downregulate the expression of GPX4 protein, thereby inducing ferroptosis in tumor cells.

[0058] Example 12 Glucose content determination: 4T1 cells were fed at a rate of 2 × 10 5Cells were seeded at a density of / well in 6-well plates and cultured overnight at 37°C and 5% CO2. Cells were then incubated for another 24 h in medium containing PTX, VE, VEP, VEP@HA, or VEP@HA-NO (50 μg / mL). The cell culture supernatant from each group was collected, and glucose levels were determined according to the Glu assay kit instructions.

[0059] The results are as follows Figure 8 As shown, compared with the control group and the PTX group, the glucose content in the culture supernatant of cells treated with VE, VEP, VEP@HA, and VEP@HA-NO was significantly increased, indicating that the above agents could effectively inhibit cellular glucose uptake. This is mainly attributed to the inhibitory effect of EGCG on key enzymes of glucose metabolism, thereby blocking the energy supply pathway of tumor cells. Among them, VEP@HA-NO had the most significant inhibitory effect on glucose uptake, which may be related to its enhanced cellular uptake mediated by the CD44 receptor.

[0060] Example 13 Assessment of the actin cytoskeleton: 4T1 cells were fed at a dose of 1×10 5 Cells were seeded at a density of / wells onto 12-well cell culture slides and cultured until adherent. Medium containing PTX, VE, VEP, VEP@HA, or VEP@HA-NO (50 μg / mL) was added to each well, and incubation continued for 24 h. After incubation, the drug-containing medium was discarded, and the cells were washed three times with PBS. The cells were then fixed sequentially with 4% paraformaldehyde for 15 min, infiltrated with 0.1% Triton X-100 for 10 min, blocked with 5% goat serum for 30 min, incubated with Actin-Tracker Green-488 in the dark for 1 h, and stained with DAPI for 5 min. The structural changes of actin were then observed using a confocal microscope.

[0061] Actin, a core component of the cytoskeleton, plays a crucial role in maintaining cell morphology, motility, and intracellular transport. To assess the effects of different treatments on cytoskeleton integrity, Actin-Tracker Green-488 staining was used to observe actin structure. The results are as follows: Figure 9 As shown, the cytoskeleton in the PTX-treated group did not show significant changes, while the intensity of green fluorescence in the cells of the VE and VEP groups was reduced, indicating damage to some cell structures. VEP@HA and VEP@HA-NO treatments induced widespread cytoskeleton collapse, manifested as cell contraction and abnormal actin aggregation. This morphological characteristic is consistent with the cytoskeleton disruption caused by disulfide bond breakage during disulfide death, demonstrating that VEP@HA-NO can induce disulfide death.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanomaterial for regulating sugar metabolism, characterized in that, The aforementioned sugar metabolism regulating nanomaterials include a metal-phenolic network core, a chemotherapeutic drug loaded in the metal-phenolic network core, and a nitric oxide donor conjugate modified on the surface of the metal-phenolic network core.

2. The glucose metabolism regulating nanomaterial as described in claim 1, characterized in that, The nitric oxide donor conjugate is nitrosylated N-acetylcysteine-hyaluronic acid.

3. The glucose metabolism regulating nanomaterial as described in claim 1, characterized in that, The metal-phenolic network core is formed by coordination of a metal salt and a polyphenolic compound. Preferably, the metal salt includes one of ammonium metavanadate, sodium orthovanadate, vanadium oxysulfate, or vanadium chloride; and the polyphenolic compound includes one of tannic acid, gallic acid, or EGCG.

4. The glucose metabolism regulating nanomaterial as described in claim 1, characterized in that, The chemotherapy drugs include at least one of paclitaxel, docetaxel, doxorubicin, or cyclophosphamide.

5. A method for preparing the sugar metabolism regulating nanomaterial according to any one of claims 1-4, characterized in that, Includes the following steps: N-acetylcysteine ​​was added to an aqueous solution of hydrochloric acid containing sodium nitrite to react and obtain nitrosylated N-acetylcysteine. The nitrosylated N-acetylcysteine ​​was then activated by carboxylation and added dropwise to hyaluronic acid to prepare nitrosylated N-acetylcysteine-hyaluronic acid. Chemotherapy drug solution, metal salt solution and nitrosylated N-acetylcysteine-hyaluronic acid solution were stirred and mixed, and polyphenol compound solution was added to react. After dialysis and freeze-drying, sugar metabolism regulating nanomaterials were obtained.

6. The preparation method according to claim 5, characterized in that, The stirring reaction time is 6~36 h, and the stirring speed is 200~1000 rpm; Alternatively, the dialysis may specifically involve dialysis for 1 to 3 days using a dialysis bag with a molecular weight cutoff of 1000-10000 Da, followed by freeze-drying for 24 to 48 hours. Alternatively, carboxyl activation specifically involves activating the carboxyl group of nitrosylated N-acetylcysteine ​​using dicyclohexylcarbodiimide and 4-dimethylaminopyridine.

7. The preparation method according to claim 5, characterized in that, The molar ratio of the metal salt to the polyphenol compound is 1:(0.3~3); The molar ratio of the nitrosylated N-acetylcysteine ​​to hyaluronic acid is 1:(0.5~2).

8. The preparation method according to claim 5, characterized in that, The mass ratio of the metal salt, polyphenol compound, chemotherapeutic drug and nitrosylated N-acetylcysteine-hyaluronic acid is 1:(1~5):(0.5~2):(1~3).

9. The use of a glucose metabolism regulating nanomaterial according to any one of claims 1-4, or a glucose metabolism regulating nanomaterial prepared by the preparation method of the glucose metabolism regulating nanomaterial according to any one of claims 5-8, in the preparation of an antitumor drug, wherein the drug comprises at least one or more of the following uses: 1) It inhibits glucose metabolism, blocks glucose uptake, disrupts the actin cytoskeleton, and induces disulfide death; 2) Consuming GSH downregulates GPX4 protein expression, inducing ferroptosis; 3) It produces NO, which promotes drug penetration and inhibits cell metastasis; 4) Reverse the immunosuppressive tumor microenvironment and improve anti-tumor efficacy.

10. An antitumor drug composition, characterized in that, The pharmaceutical composition comprises the glucose metabolism regulating nanomaterials as described in any one of claims 1-4 and a pharmaceutically acceptable carrier; the dosage form of the pharmaceutical composition is at least one of an injection, a nanoparticle formulation, or a lyophilized powder injection.

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