Albumin nanoparticle co-loading with menadione and lactate oxidase, preparation method and application

Abstract

This invention discloses albumin nanoparticles co-loaded with flavin and lactate oxidase, their preparation method, and applications. Using human serum albumin as a carrier, flavin and lactate oxidase are simultaneously encapsulated at a mass ratio of 10:1, with a particle size of 150-180 nm and a polydispersity index of less than 0.2. This invention exerts its anti-hepatocellular carcinoma effect through mechanisms such as reducing lactate levels in the tumor microenvironment, inducing an increase in reactive oxygen species, inhibiting cGAS protein lactation modification, and activating the cGAS-STING signaling pathway. The nanoparticles exhibit high encapsulation efficiency and good stability. The flavin loaded on these nanoparticles can significantly reduce the reduction of pyruvate to lactate by inhibiting lactate dehydrogenase; simultaneously, the lactate oxidase loaded on these nanoparticles catalyzes the dehydrogenation of lactate to pyruvate. In other words, these nanoparticles synergistically regulate lactate metabolism through a two-way regulatory strategy of "reducing source and increasing sink"—"inhibiting lactate production" and "promoting lactate clearance"—significantly enhancing in vitro and in vivo anti-tumor effects, and demonstrating high biosafety.

Description

Technical Field

[0001] This invention relates to the fields of biomedical technology and nanomedicine delivery, specifically to a human serum albumin nanoparticle co-loaded with galloflavin (GAL) and lactate oxidase (LOD), its preparation method, and its application in the treatment of hepatocellular carcinoma. Background Technology

[0002] Liver cancer is one of the most common malignant tumors worldwide, with hepatocellular carcinoma (HCC) accounting for a very high proportion. Its highly invasive nature and drug resistance pose a significant challenge to clinical treatment. In China, there are 389,000 new cases of liver cancer and 336,000 deaths annually, ranking second among malignant tumor deaths and seriously threatening public health. Traditional anti-liver cancer drugs have drawbacks such as non-specific distribution and low targeted delivery efficiency, and are prone to causing serious toxic side effects. Therefore, the development of novel targeted therapy strategies is an urgent clinical need.

[0003] In the tumor microenvironment (TME), tumor cells produce large amounts of lactate through the "Warburg effect," creating a high-lactate microenvironment. Lactic acid not only provides energy for the rapid proliferation of tumor cells but also promotes tumor invasion and metastasis by lowering the microenvironment pH. Furthermore, it can activate signaling pathways such as HIF-1α and STAT3 to maintain tumor cell survival. In terms of immune regulation, lactate can inhibit the antigen-presenting function of dendritic cells (DCs), promote the polarization of macrophages towards the pro-tumorigenic M2 type, and weaken the killing ability of T cells against tumor cells, forming an immunosuppressive microenvironment and accelerating tumor immune escape.

[0004] Lactic acidification, as an emerging post-translational modification of proteins, is closely related to tumor progression. Lactic acid can bind to lysine residues of proteins via ester bonds, regulating protein function. Studies have confirmed that high concentrations of lactate can mediate cGAS protein lactation modification through aminoacyl-tRNA synthetases (AARS1 and AARS2), inhibiting its DNA binding ability, blocking cGAS-STING pathway activation, reducing interferon-β (IFN-β) secretion, and forming a vicious cycle of immune escape. This provides a new target for the treatment of liver cancer.

[0005] Intervention strategies targeting lactate metabolism mainly fall into two categories: inhibiting production and promoting clearance. GAL, a novel non-competitive lactate dehydrogenase inhibitor, can block lactate production at its source, but its strong hydrophobicity leads to low bioavailability and poor targeting. Lactate oxidase (LOD) catalyzes the breakdown of lactate into pyruvate and hydrogen peroxide, reducing lactate levels and improving tumor hypoxia; however, free LOD has poor stability, is easily degraded by proteases, and hydrogen peroxide accumulation may damage normal tissues. Existing research mostly focuses on single intervention pathways; synergistic regulatory strategies involving two pathways have not yet been systematically explored.

[0006] Albumin nanoparticles offer significant advantages as drug delivery carriers: they exhibit good biocompatibility, are non-immunogenic, and possess multiple drug-binding sites, enabling efficient loading of hydrophobic drugs through physical encapsulation or chemical bonding. They can achieve passive tumor targeting via the EPR effect and bind to gp60 receptors on tumor cell surfaces, enhancing cellular uptake through SPARC-mediated endocytosis. Furthermore, their long in vivo circulation time reduces rapid drug clearance. Based on these advantages, an albumin nanoparticle delivery system co-loaded with GAL and LOD was constructed to achieve synergistic regulation of a dual pathway of "inhibiting lactate production + accelerating lactate clearance," simultaneously addressing the delivery challenges of both drugs and providing a feasible solution for combined metabolic and immunotherapy for liver cancer.

[0007] Definitions: GAL: Galloflavin, a lactate dehydrogenase inhibitor.

[0008] LOD: Lactate Oxidase, an enzyme that catalyzes the breakdown of lactate.

[0009] HSA: Human Serum Albumin.

[0010] GAL / LOD@HSA NPs: albumin nanoparticles co-loaded with flavin and lactate oxidase.

[0011] HCC: Hepatocellular Carcinoma.

[0012] PDI: Polydispersity Index, used to measure the uniformity of particle size distribution.

[0013] TGI: Tumor Growth Inhibition. Summary of the Invention

[0014] The purpose of this invention is to provide a method for preparing albumin nanoparticles co-loaded with flavin and lactate oxidase and their application, so as to solve the problems of poor effect and low drug delivery efficiency of single lactate regulation strategy in the prior art.

[0015] To achieve the above-mentioned objectives, the present invention provides the following technical solution: An albumin nanoparticle co-loaded with flavin and lactate oxidase, wherein the albumin nanoparticle co-loaded with flavin and lactate oxidase uses human serum albumin as a carrier and simultaneously encapsulates flavin and lactate oxidase.

[0016] In a further improvement, the mass ratio of the flavonoid to lactate oxidase is 10:1.

[0017] In a further improvement, the albumin nanoparticles have a particle size of 150-180 nm and a polydispersity index of less than 0.2.

[0018] A method for preparing albumin nanoparticles co-loaded with flavin and lactate oxidase as described above includes the following steps: Step 1: Dissolve human serum albumin in water and adjust the pH to alkaline to obtain an albumin solution; Step 2: Add lactate oxidase solution and chlorogenic acid solution to the albumin solution in sequence, and mix well to obtain a mixed solution; Step 3: Add a desolvating agent to the mixed solution while stirring to cause albumin to aggregate into nanoparticles; Step 4: Add a cross-linking agent to the nanoparticles to carry out a cross-linking reaction, and after purification, obtain albumin nanoparticles co-loaded with flavin and lactate oxidase.

[0019] In a further improvement, in step two, the mass ratio of flavin to lactate oxidase is 10:1; in step three, the desolvation reagent is anhydrous ethanol; and in step four, the crosslinking agent is glutaraldehyde.

[0020] In a further improvement, the purification step in step four is any one or a combination of dialysis and ultrafiltration to remove unencapsulated free drug and organic solvent.

[0021] An application of albumin nanoparticles co-loaded with flavin and lactate oxidase as described above, characterized in that the albumin nanoparticles co-loaded with flavin and lactate oxidase are used to prepare antitumor drugs.

[0022] A further improvement is that the albumin nanoparticles co-loaded with flavin and lactate oxidase are used to prepare anti-liver cancer drugs.

[0023] In a further improvement, the amount of albumin nanoparticles co-loaded with flavin and lactate oxidase is 0.5 mg / kg.

[0024] In a further improvement, the albumin nanoparticles co-loaded with flavin and lactate oxidase are used to prepare drugs that reduce lactate levels in tumor cells, induce an increase in reactive oxygen species levels in tumor cells, cause a decrease in mitochondrial membrane potential, induce tumor cell apoptosis, inhibit lactation modification of cGAS protein, activate the cGAS-STING signaling pathway, and promote IFN-β secretion.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. A high-performance co-load nanodelivery system was constructed: This invention prepares human serum albumin nanoparticles (GAL / LOD@HSA NPs) co-loaded with flavin (GAL) and lactate oxidase (LOD) via a solvent-free crosslinking method. The resulting nanoparticles have a uniform particle size (approximately 167.9 nm, PDI = 0.125) and exhibit good colloidal stability in various biological media. The nanoparticles achieve encapsulation rates of up to 81.86% for GAL and 65.35% for LOD, effectively maintaining the catalytic activity of LOD and overcoming the drawbacks of GAL's strong hydrophobicity and low bioavailability, as well as LOD's poor stability and easy degradation.

[0026] 2. The optimal drug synergy ratio was determined: This invention, through the MTT assay combined with Chou-Talalay co-index analysis, determined the optimal synergistic mass ratio of GAL to LOD to be 10:1. At this ratio, the synergistic effect of the two drugs is strongest, laying the foundation for achieving synergistic regulation of both "inhibition of lactate production" and "acceleration of lactate clearance" through dual pathways.

[0027] 3. Exhibits excellent in vitro antitumor activity: Cellular uptake experiments demonstrated that GAL / LOD@HSA NPs were efficiently and time-dependently taken up by Hepa1-6 liver cancer cells. In vitro cytotoxicity experiments showed that the nanoparticles exhibited dose-dependent cytotoxicity against Hepa1-6 cells, with a half-maximal inhibitory concentration (IC50) of [missing value]. The drug efficacy was significantly lower than that of the free single-drug and combination drug groups; the live and dead cell staining results further and intuitively confirmed its powerful killing effect. This fully demonstrates that the nanodelivery system constructed in this invention can significantly enhance the antitumor efficacy of drugs.

[0028] 4. Demonstrates potent in vivo antitumor effects and good biocompatibility: In the Hepa1-6 tumor-bearing mouse model, GAL / LOD@HSA NPs, administered via tail vein injection, effectively inhibited tumor growth, achieving a tumor growth inhibition rate (TGI) of up to 85.4%, significantly superior to the single-drug-loaded nanoparticle group (see [link to relevant documentation]). Figure 11a, 11b). No significant decrease in body weight was observed in any group of mice during treatment, indicating that the nanoparticle system has low toxicity and good safety (see [reference]). Figure 11 c). H&E staining results of tumor tissue showed that the tumor cells in the GAL / LOD@HSA NPs treatment group exhibited nuclear condensation and fragmentation, displaying the most obvious tissue necrosis characteristics (see [link]). Figure 11 (d) further confirms its powerful in vivo anti-tumor effect.

[0029] 5. In vitro cell experiments demonstrated that GAL / LOD@HSA NPs can be efficiently taken up by liver cancer cells, dose-dependently inhibiting cell proliferation and effectively inducing apoptosis in liver cancer cells by increasing intracellular ROS and decreasing mitochondrial membrane potential. More importantly, these nanoparticles can significantly reduce intracellular lactate levels, inhibit lactation modification of cGAS protein, thereby restoring cGAS activity, activating the downstream STING signaling pathway, and promoting the secretion of the immunostimulatory factor IFN-β. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the preparation of the GAL / LOD@HSA NPs of the present invention.

[0031] Figure 2 This is a graph showing the screening results for the optimal synergistic ratio of GAL and LOD in this invention. (a) Cell viability after treatment with different drug ratios; (b) Synergistic index of different drug ratios.

[0032] Figure 3 The following figures represent the characterization results of the GAL / LOD@HSA NPs of this invention. (a) Particle size distribution determined by dynamic light scattering; (b) Transmission electron microscopy image; (c) Particle size variation in different media; (d) Particle size variation after 7 days of storage in PBS.

[0033] Figure 4 The drug loading performance of the GAL / LOD@HSA NPs of this invention is shown in the figures: (a) UV curves of different concentrations of GAL; (b) standard curve of GAL; (c) UV curve of free GAL; (d) standard curve of LOD. Figure 5 The level of H2O2 after LOD and GAL / LOD@HSA NPs treatment according to the present invention.

[0034] Figure 6 This invention illustrates the uptake of GAL / LOD / RhB@HSA NPs by Hepal-6 cells. (a) Observation of GAL / LOD / RhB@HSA NPs uptake by Hepal-6 cells using an inverted microscope; (b) Detection of GAL / LOD / RhB@HSA NPs uptake by Hepal-6 cells using flow cytometry; (c) Statistical graph of average fluorescence intensity from flow cytometry results.

[0035] Figure 7 Survival rates of Hepal-6 cells after treatment with different drugs.

[0036] Figure 8 AM / PI staining of Hepal-6 cells after different drug treatments.

[0037] Figure 9 To detect the lactation level of total protein in Hepa1-6 cells after different drug treatments. (a) Western blotting (WB) to detect the lactation level of total protein; (b) Quantification of WB results by grayscale values. ***p<0.001, **p<0.01, *p<0.05.

[0038] Figure 10 The cGAS lactation level in Hepa1-6 cells after different drug treatments. (a) Western blot analysis of cGAS lactation level; (b) cGAS protein expression level; (c) grayscale quantification of cGAS protein expression level. ***p<0.001, **p<0.01, *p<0.05.

[0039] Figure 11 To assess the in vivo antitumor activity of GAL / LOD@HSA NPs. (a) Stereoscopic tumor morphology images of all groups; (b) Tumor volume of Hepal-6 tumor-bearing mice; (c) Body weight change curves of Hepal-6 tumor-bearing mice; (d) HE staining images of tumor tissues after different drug treatments. Detailed Implementation Example 1:

[0040] Preparation and characterization of albumin nanoparticles co-loaded with flavin and lactate oxidase (GAL / LOD@HSA NPs):

[0041] Preparation method: GAL / LOD@HSA NPs were prepared using a desolvation crosslinking method. First, human serum albumin (HSA) was dissolved in deionized water to prepare a 10 mg / mL solution, and the pH was adjusted to 8.5 with sodium hydroxide. In a 25 mL round-bottom flask, 5 mL of the above HSA solution, 50 μL of a 10 mg / mL lactate oxidase (LOD) solution, and 500 μL of a 5 mg / mL glycopyrrolidone (GAL) solution were added sequentially and mixed thoroughly (GAL to LOD mass ratio was 10:1). While continuously stirring, 5 mL of anhydrous ethanol was slowly added dropwise as a desolvation agent to allow albumin to aggregate into nanoparticles. Subsequently, 100 μL of 5% glutaraldehyde solution was added, and the crosslinking reaction was carried out at room temperature for 2 hours. The reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 100 kDa and dialyzed against deionized water for 24 hours to remove organic solvents and unencapsulated drug. Finally, glucose was added as a lyophilization protectant, and after lyophilization, powdered GAL / LOD@HSA NPs were obtained. A schematic diagram of the construction of this invention is shown below. Figure 1 As shown.

[0042] Particle size and morphology characterization: An appropriate amount of GAL / LOD@HSA NPs was dispersed in deionized water, and its hydrated particle size and polydispersity index were determined using a dynamic light scattering particle size analyzer. Results are as follows: Figure 3 As shown in Figure a, the average particle size of the nanoparticles is 167.9 nm, and the polydispersity index (PDI) is 0.125, indicating a uniform particle size distribution. The morphology of the nanoparticles was observed using a transmission electron microscope, as shown in Figure a. Figure 3 As shown in b, the nanoparticles are regularly spherical and well dispersible. Furthermore, dispersing the nanoparticles in different media (water, physiological saline, DMEM containing 10% FBS, and 1640 medium) did not show significant changes in particle size. Figure 3 c); The particle size remained stable after 7 days of storage in PBS (pH 7.4). Figure 3 d), indicating that the nanoparticles prepared in this invention have good colloidal stability.

[0043] Encapsulation efficiency determination: The encapsulation efficiency of GAL was determined by ultraviolet spectrophotometry. First, a standard curve for GAL at 385 nm was established. Figure 4 a, 4b). The GAL / LOD@HSA NPs solution was centrifuged in a 3 kDa ultrafiltration centrifuge tube, the filtrate was collected, and the absorbance of free GAL was measured ( Figure 4 c), the encapsulation efficiency was calculated to be 81.86%. The encapsulation efficiency of LOD was determined using the Bradford method, and a standard curve for LOD was established. Figure 4d) The nanoparticle solution was centrifuged in a 50 kDa ultrafiltration centrifuge tube, and the filtrate was collected to determine the free LOD concentration. The encapsulation efficiency was calculated to be 65.35%. The results show that the nanoparticles prepared in this invention have high encapsulation efficiency for both GAL and LOD.

[0044] Enzyme activity assay: To verify the activity of LOD in the nanoparticles, an H2O2 content detection kit was used. GAL / LOD@HSA NPs and free LOD were added to a solution containing 10 mM lactic acid, and the H2O2 content generated at different time points was measured. The results are as follows: Figure 5 As shown, the amount of H2O2 generated in both groups increased with time, and the GAL / LOD@HSA NPs group showed a gradual upward trend, reaching a plateau after 2 hours, indicating that the LOD in the nanoparticles retained good catalytic activity and could achieve stable release. Example 2: Screening for the optimal synergistic ratio of flavin and lactate oxidase

[0045] The MTT assay was used to investigate the synergistic antiproliferative effect of GAL and LOD on Hepa1-6 cells. Logarithmically growing Hepa1-6 cells were seeded at 8000 cells per well in 96-well plates and cultured for 24 hours. Different concentrations of GAL, LOD alone, and different ratios (LOD:GAL = 1:1, 1:10, 1:30) of the combined drug were then added, and incubation continued for another 24 hours. Cell viability was assessed using the MTT assay, and the combination index (CI) was calculated using the Chou-Talalay method. Results are shown below. Figure 2 As shown in figure a, different proportions of drug combinations all exhibited concentration-dependent cytotoxicity. This was determined by calculating the CI value (…). Figure 2 (b) It was found that when LOD:GAL = 1:10, the CI value was 0.7161, which is less than 1, indicating the strongest synergistic effect. Therefore, the optimal synergistic mass ratio of GAL to LOD was determined to be 10:1 for subsequent nanoparticle preparation. Example 3: Cell uptake experiment:

[0046] To investigate the cellular uptake capacity of the nanoparticles, fluorescent nanoparticles GAL / LOD / RhB@HSA NPs loaded with Rhodamine B (RhB) were prepared according to the method in Example 1. Hepa1-6 cells were seeded in 12-well plates and cultured overnight. Then, GAL / LOD / RhB@HSA NPs (2 μg / mL based on LOD concentration) were added, and the cells were incubated for 1, 2, 4, and 6 hours, respectively. After washing the cells with PBS, the nuclei were stained with Hoechst 33342, and observed using an inverted fluorescence microscope. Figure 6As shown in Figure a, the intracellular red fluorescence (RhB) gradually increased with prolonged incubation time, indicating that the nanoparticles were taken up by the cells in a time-dependent manner. Further quantitative analysis using flow cytometry also showed that the fluorescence intensity increased with time. Figure 6 b), the average fluorescence intensity reached its highest level after 6 hours of incubation. Figure 6 c) indicates that the nanoparticles reach saturation after approximately 6 hours. These results confirm that the GAL / LOD@HSA NPs prepared in this invention can be efficiently taken up by liver cancer cells. Example 4: In vitro cytotoxicity evaluation:

[0047] The in vitro antitumor activity of GAL / LOD@HSA NPs against Hepa1-6 cells was evaluated using the MTT assay and Calcein-AM / PI live / dead cell staining assay.

[0048] MTT assay: Hepa1-6 cells were seeded at 8000 cells per well in 96-well plates and cultured for 24 hours. Different concentrations of free GAL, free LOD, LOD+GAL (1:10) mixture, and GAL / LOD@HSA NPs (based on final LOD concentration) were then added, and the cells were cultured for another 24 hours. Cell viability was assessed using the MTT assay, and the half-maximal inhibitory concentration (IC50) was calculated. The result is as follows: Figure 7 As shown, GAL / LOD@HSA NPs The value (1.362 μg / mL) was significantly lower than that of the LOD+GAL (1:10) mixture (2.259 μg / mL), indicating that the nanoparticles were more cytotoxic, which may be due to the improved intracellular drug delivery efficiency of the nanoparticles.

[0049] Live and dead cell staining: Hepa1-6 cells were seeded into 6-well plates and cultured for 24 hours. Then, they were treated for 24 hours with PBS, free GAL, free LOD, a mixture of LOD and GAL (1:10), and GAL / LOD@HSA NPs, respectively. Subsequently, they were stained with Calcein-AM (for live cells, green) and PI (for dead cells, red) and observed under a fluorescence microscope. Figure 8 As shown, almost all cells in the control group and HSA NPs group exhibited green fluorescence, indicating good cell survival; a small amount of red fluorescence was observed in the free drug group; while the GAL / LOD@HSA NPs treatment group showed the strongest red fluorescence, indicating a large number of cell deaths. This result is consistent with the MTT results, further confirming that the GAL / LOD@HSA NPs prepared in this invention have excellent in vitro antitumor effects. Example 5: Detection of cellular lactation levels and cGAS-STING signaling pathway

[0050] 5.1 Detection of total protein lactation level in cells: Logarithmically growing Hepa1-6 cells were seeded into culture plates. After adhesion, physiological saline, free GAL, free LOD, a GAL+LOD (10:1) mixture, and GAL / LOD@HSA NPs were added, and the cells were cultured for another 24 h. The culture medium was discarded, and the cells were washed three times with pre-cooled PBS. RIPA lysis buffer containing PMSF and a phosphatase inhibitor was added, and the cells were lysed on ice for 30 min. The cells were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected to obtain total protein. After quantification using a BCA kit, SDS-PAGE electrophoresis was performed, and the cells were transferred to a PVDF membrane and blocked with 5% skim milk powder at room temperature for 2 h. Anti-lactation antibody was added and incubated overnight at 4 °C. After washing with TBST, secondary antibody was added and incubated at room temperature for 2 h. ECL chemiluminescence was used for color development, and the bands were acquired and analyzed using a gel imaging system.

[0051] Experimental results: such as Figure 9 As shown, compared with the control group, all drug-treated groups can reduce the level of total cellular protein lactation. Among them, the GAL / LOD@HSA NPs group has the most significant inhibitory effect and can effectively reduce the overall cellular lactation modification level.

[0052] 5.2 Detection of cGAS protein lactation level and expression: 500 μg of the total protein was taken, and 2 μL of cGAS antibody was added. An IgG negative control was also included. The mixture was incubated overnight at 4°C with a shaker. Protein A / G magnetic beads were washed three times, and a protein-antibody mixture was added and incubated at 4°C for 4 h. After washing and separating the magnetic beads, 2×SDS loading buffer was added, and the protein was eluted by boiling in a water bath for 10 min. Western blotting was used to detect the cGAS lactation level and total cGAS protein expression.

[0053] Experimental results: such as Figure 10 As shown, GAL / LOD@HSA NPs can significantly reduce the lactation modification level of cGAS protein and upregulate the expression of total cGAS protein. The effect is significantly better than that of free single drug and combination drug group, effectively relieving the inhibition of cGAS protein by lactate. Example 6: In vivo antitumor pharmacodynamic study of GAL / LOD@HSA NPs:

[0054] A subcutaneous tumor-bearing C57BL / 6 mouse model of Hepa1-6 cells was established to evaluate the in vivo antitumor effect of GAL / LOD@HSA NPs.

[0055] Animal model construction: Log-phase murine Hepa1-6 liver cancer cells were obtained, and the cell suspension concentration was adjusted to... A liver cancer xenograft model was constructed by subcutaneously injecting 100 μL of cell suspension into the right axilla of 4-week-old male C57 mice.

[0056] Grouping and Administration: When the tumor volume reached approximately 100 mm³, tumor-bearing mice were randomly divided into 5 groups (n≥5) using a simple random sampling method: saline control group, HSA NPs group, GAL@HSA NPs group, LOD@HSA NPs group, and GAL / LOD@HSA NPs group. The dosage for each group was 0.5 mg / kg (LOD), administered via tail vein injection every other day for 14 consecutive days. Tumor volume and mouse weight were measured every other day during treatment. The tumor volume was calculated using the formula: Tumor volume = (Tumor length × Tumor width²) / 2. After treatment, mice were sacrificed, tumor tissue was dissected, weighed, and photographed to calculate the tumor growth inhibition rate (TGI). The tumor tissue was fixed with 4% paraformaldehyde and stained with hematoxylin and eosin (H&E) to observe histopathological changes.

[0057] Experimental Results: After treatment, the morphological images of ex vivo tumors in each group were as follows: Figure 11 As shown in Figure a, the GAL / LOD@HSA NPs group had the smallest tumor volume. Tumor volume growth curve ( Figure 11 (b) shows that the tumors in the control group exhibited a rapid growth trend, while all treatment groups showed varying degrees of tumor suppression effects. The GAL / LOD@HSA NPs group showed the most significant tumor suppression effect, with a tumor growth inhibition rate (TGI) as high as 85.4%. Mouse body weight change curve ( Figure 11 c) shows that no significant decrease in body weight was observed in any group of mice during treatment, indicating that the nanoparticle system has good in vivo safety. Tumor tissue H&E staining results ( Figure 11 d) The results showed that the tumor cells in the GAL / LOD@HSA NPs group underwent nuclear condensation and fragmentation, exhibiting the most obvious tissue necrosis characteristics, further confirming its powerful in vivo anti-tumor effect.

Claims

1. An albumin nanoparticle co-loaded with flavin and lactate oxidase, characterized in that, The albumin nanoparticles co-loaded with flavin and lactate oxidase use human serum albumin as a carrier and simultaneously encapsulate flavin and lactate oxidase.

2. The albumin nanoparticles co-loaded with flavin and lactate oxidase according to claim 1, characterized in that, The mass ratio of flavin to lactate oxidase is 10:

1.

3. The albumin nanoparticles co-loaded with flavin and lactate oxidase according to claim 1 or 2, characterized in that, The albumin nanoparticles have a particle size of 150-180 nm and a polydispersity index of less than 0.

2.

4. A method for preparing albumin nanoparticles co-loaded with flavin and lactate oxidase as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1: Dissolve human serum albumin in water and adjust the pH to alkaline to obtain an albumin solution; Step 2: Add lactate oxidase solution and chlorogenic acid solution to the albumin solution in sequence, and mix well to obtain a mixed solution; Step 3: Add a desolvating agent to the mixed solution while stirring to cause albumin to aggregate into nanoparticles; Step 4: Add a cross-linking agent to the nanoparticles to carry out a cross-linking reaction, and after purification, obtain albumin nanoparticles co-loaded with flavin and lactate oxidase.

5. The method for preparing albumin nanoparticles co-loaded with flavin and lactate oxidase according to claim 4, characterized in that, In step two, the mass ratio of flavin to lactate oxidase is 10:1; in step three, the desolvation reagent is anhydrous ethanol; and in step four, the crosslinking agent is glutaraldehyde.

6. The method for preparing albumin nanoparticles co-loaded with flavin and lactate oxidase according to claim 4, characterized in that, The purification step in step four is any one or a combination of dialysis and ultrafiltration to remove unencapsulated free drug and organic solvent.

7. The application of albumin nanoparticles co-loaded with flavin and lactate oxidase as described in claim 1 or 2, characterized in that, The albumin nanoparticles co-loaded with flavin and lactate oxidase are used to prepare antitumor drugs.

8. The application of albumin nanoparticles co-loaded with flavin and lactate oxidase as described in claim 7, characterized in that, The albumin nanoparticles co-loaded with flavin and lactate oxidase are used to prepare anti-liver cancer drugs.

9. The application of albumin nanoparticles co-loaded with flavin and lactate oxidase according to claim 7, characterized in that, The amount of albumin nanoparticles co-loaded with flavin and lactate oxidase is 0.5 mg / kg.

10. The application of albumin nanoparticles co-loaded with flavin and lactate oxidase according to claim 7, characterized in that, The albumin nanoparticles co-loaded with flavin and lactate oxidase are used to prepare drugs that reduce intracellular lactate levels in tumor cells, induce an increase in intracellular reactive oxygen species levels, cause a decrease in mitochondrial membrane potential, induce tumor cell apoptosis, inhibit lactation modification of cGAS protein, activate the cGAS-STING signaling pathway, and promote IFN-β secretion.

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