A lipoxygenase inhibitor and its preparation and use

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a lecithin nanomaterial carrying oxamic acid salt and a preparation method and application thereof. The lecithin nanomaterial carrying oxamic acid salt (OXA) has a shell-core structure; the shell is a phospholipid bilayer structure formed by soybean lecithin and DSPE-PEG2000; and the core is oxamic acid salt. The lecithin nanomaterial LEC (OXA) carrying oxamic acid salt in the application uses natural soybean lecithin as a carrier, improves the defects of poor physical and chemical properties and low stability of the natural lecithin-based nanomaterial, solves the problems of high polarity, weak membrane penetration and low in-vivo bioavailability of OXA, realizes the synergistic effect of the anticancer performance of soybean lecithin and the lactic acid generation inhibition of OXA, can target to reduce the lactic acid concentration of tumors, reverse the DC-T cell immunosuppression of residual cancer microenvironment, has the tumor-targeting immunoregulation and anticancer performance, and provides a new drug carrier and treatment strategy for the clinical combined immunotherapy of hepatocellular carcinoma RFA postoperative residual cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a lecithin nanomaterial loaded with oxalate salt, its preparation method, and its application. Background Technology

[0002] Radiofrequency ablation (RFA) is currently the first-line treatment recommended by clinical guidelines for early-stage liver cancer. However, in practice, some patients, due to factors such as large lesion size or proximity to vital organs or blood vessels, often cannot achieve complete ablation, leading to incomplete radiofrequency ablation (IRFA). Multiple studies have confirmed that residual tumor tissue after IRFA is prone to rapid proliferation and invasion, significantly increasing the risk of recurrence and ultimately negatively impacting prognosis.

[0003] From an immune mechanism perspective, during radiofrequency ablation, tumor tissue releases damage-associated molecular patterns and tumor-associated antigens, activating the cGAS-STING-IRF3 pathway and promoting the expression of type I interferon and interferon-stimulated genes (ISGs). Theoretically, ISGs can recruit immune cells, enhance dendritic cell (DC) activation, and promote CD8 expression. + T-cell killing activity eliminates residual cancer cells. However, in reality, DC-CD8 in residual cancer cells after IRFA surgery... + T-cell anti-tumor immunity was not effectively activated, and the immune response induced by ablation itself was insufficient to effectively eliminate residual cancer cells. Its regulatory mechanism is not yet clear, and it cannot solve the problem of residual cancer cell recurrence.

[0004] Current intervention strategies for residual cancer after IRFA surgery have significant shortcomings: First, the specific mediating mechanism of immunosuppression in residual cancer is unclear, making it impossible to locate key targets regulating immune resistance and lacking specific reversal methods. Second, existing drugs are significantly inadequate; conventional chemotherapy drugs have poor targeting, strong toxic side effects, limited efficacy, and are prone to inducing drug resistance. Existing targeted drugs are designed for the overall pathogenesis of liver cancer but are not adapted to the special immune microenvironment of residual cancer, and cannot effectively reverse immune resistance. Third, basic research lacks treatment regimens for residual cancer after IRFA surgery in mouse liver cancer orthotopic tumors, especially strategies that can synergize with immune checkpoint inhibitors such as PD-1 antibodies and have clinical translational potential, leading to a disconnect between basic and clinical research and failing to meet clinical needs.

[0005] In summary, the difficulty in eliminating residual cancer cells after IRFA surgery is a key factor leading to patient recurrence and poor treatment outcomes. Current technologies and drugs cannot address this problem due to the lack of a clear immunosuppressive mechanism, effective targets, and targeted agents. Therefore, there is an urgent need to provide a technical solution that clarifies the immunosuppressive mechanism of residual cancer cells, screens effective targets that can reverse immune resistance, and develops targeted drugs. This would provide theoretical support and translational strategies for clinical immunotherapy, fill technological gaps, and meet clinical needs. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide a lecithin nanomaterial loaded with oxalate, its preparation method and application, in order to solve the problem of the lack of effective treatment options in the intervention strategies for residual cancer after IRFA surgery in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] This invention provides a lecithin nanomaterial loaded with oxalate, wherein the lecithin nanomaterial loaded with oxalate has a shell-core structure; the shell is a phospholipid bilayer structure formed by soybean lecithin and DSPE-PEG2000; and the core is oxalate.

[0009] The residual tumor microenvironment after IRFA surgery is hypoxic. Under this condition, tumor cells primarily rely on glycolysis for energy, leading to a significant increase in lactate production. Lactate may be a crucial factor mediating immunosuppression in the residual tumor microenvironment, particularly affecting dendritic cell (DC)-mediated T-cell anti-tumor immune function. The key rate-limiting enzyme for lactate production is lactate dehydrogenase A (LDHA). Currently, very few LDHA inhibitors are in clinical use. Among numerous preclinical drugs, oxalate (OXA) has been extensively explored. However, oxalate has significant drawbacks. Its high polarity makes it difficult to penetrate cell membranes, requiring high working concentrations. Even after entering the body, it is difficult to maintain effective blood drug concentrations. This low bioavailability severely limits its biological activity and pharmacological effects, becoming a key technical bottleneck in the treatment of residual tumors after IRFA surgery.

[0010] This invention first clearly elucidates the mediating effect of increased lactate in the residual cancer microenvironment after IRFA surgery on DC-CD8. +The specific molecular mechanism of T-cell immunosuppression was investigated, and lactate was identified as a key target for reversing residual cancer immune resistance, providing core theoretical support for the design of subsequent treatment plans. Based on this, the present invention further optimizes the preparation process of natural soybean lecithin, improving the physicochemical properties and stability of the prepared nanomaterials. Soybean lecithin is used as a natural carrier to encapsulate oxalate (OXA), constructing oxalate-encapsulated soybean lecithin nanomaterials LEC (OXA). The construction of this nanomaterial relies on the natural properties of soybean lecithin, which can spontaneously assemble into a phospholipid bilayer membrane, thereby stably encapsulating water-soluble oxalate to form a shell-core structure. Simultaneously, DSPE-PEG2000 is introduced, whose PEG chain significantly enhances the hydrophilicity and stability of the material, synergistically working with soybean lecithin to achieve efficient encapsulation and delivery of OXA, effectively improving the in vivo bioavailability of OXA, and simultaneously exerting the synergistic anti-cancer effect of soybean lecithin and OXA.

[0011] To verify the effectiveness of this approach, this invention applied LEC (OXA) nanomaterials to the treatment of residual cancer after IRFA surgery in mouse hepatocellular carcinoma orthotopic tumors. By inhibiting lactate production in the residual cancer tissue and reversing the immunosuppressive microenvironment, and simultaneously combining it with PD-1 antibody for synergistic immune intervention, the therapeutic effect and synergistic mechanism of this approach on residual cancer after IRFA surgery were clearly verified.

[0012] This invention also discloses a method for preparing the above-mentioned lecithin nanomaterials loaded with oxalate, comprising the following steps: Soybean lecithin and DSPE-PEG2000 were dissolved separately in organic solvents, and then mixed to obtain a mixture; the mixture was rotary evaporated until a lipid film appeared. The oxalate salt is mixed with the lipid film, and the lipid film is hydrated to form an emulsion; The emulsion was purified by sonication, ultrafiltration centrifugation, and washing with PBS. The supernatant was lecithin nanomaterials loaded with oxalate.

[0013] In some embodiments of the present invention, the mass ratio of soybean lecithin, DSPE-PEG2000, and oxalate is 1.5~2.2:0.1~0.3:60~65. Excessive soybean lecithin will cause a significant increase in particle size, forming flocculent precipitates; insufficient lecithin will lead to a decrease in drug loading. Excessive DSPE-PEG2000 will increase the polarity of the formed nanomaterials, hindering cellular uptake; insufficient DSPE-PEG2000 will lead to decreased stability and easy aggregation. Excessive oxalate will result in a large amount of free drug in the system, making it difficult to purify the material and achieve tumor-targeting effects; insufficient oxalate will result in insufficient drug loading of the nanomaterials, requiring increased intravenous dosage and affecting efficacy. Preferably, the mass ratio of soybean lecithin, DSPE-PEG2000, and oxalate is 1.5:0.1:60, 2:0.2:62.5, or 2.2:0.3:65.

[0014] In some preferred embodiments of the present invention, the mass ratio of soybean lecithin, DSPE-PEG2000 and oxalate is 2:0.2:62.5.

[0015] In some embodiments of the present invention, the organic solvent includes one or both of chloroform and methanol.

[0016] In some preferred embodiments of the present invention, the soybean lecithin has high solubility in chloroform, dissolves completely, and is easy to remove by subsequent evaporation. Therefore, using chloroform to dissolve soybean lecithin facilitates subsequent film formation.

[0017] In some preferred embodiments of the present invention, methanol is used to dissolve the DSPE-PEG2000, which makes the DSPE-PEG2000 easy to store.

[0018] In some embodiments of the present invention, after mixing the oxalate salt with the lipid film, the mixture is stirred at 1000-1400 rpm / min for 0.8-1.2 h to form an emulsion. Preferably, the stirring speed is 1000 rpm / min, 1200 rpm / min, 1300 rpm / min or 1400 rpm / min, and the stirring time is 0.8 h, 0.9 h, 1.0 h or 1.2 h.

[0019] In some embodiments of the present invention, the ultrasonic power of the emulsion is 150W~170W, and the duration is 18~23s. Excessive ultrasonic power or duration will damage the nanomaterial membrane structure, leading to drug release; insufficient power will prevent the nanomaterials from dispersing, making them prone to aggregation and increasing particle size. Preferably, the ultrasonic power can be selected as 150W, 160W, or 170W, corresponding to ultrasonic times of 23s, 20s, and 18s respectively, i.e., 150W for 23s, 160W for 20s, and 170W for 18s.

[0020] In some preferred embodiments of the present invention, the power of the ultrasound is 160W and the duration is 20s.

[0021] This invention also discloses the application of the oxaloacetate-loaded lecithin nanomaterials or the oxaloacetate-loaded lecithin nanomaterials prepared by the aforementioned method in the preparation of drugs for treating residual tumors after radiofrequency ablation of cancer.

[0022] In some embodiments of the present invention, the cancer includes liver cancer.

[0023] In some embodiments of the present invention, lecithin nanomaterials loaded with oxalate are used for targeted treatment of residual cancer cells in situ after IRFA surgery in mice with liver cancer.

[0024] In some embodiments of the present invention, the drug comprises a combination of lecithin nanomaterials loaded with oxalate and PD-1 antibody.

[0025] This invention also discloses a drug for treating residual tumors after radiofrequency ablation of cancer, the lecithin nanomaterial loaded with oxalate, and a pharmaceutically acceptable carrier.

[0026] Based on the technical solution of the present invention, the present invention has the following beneficial effects compared with the prior art: The oxaloacetate-loaded lecithin nanomaterial LEC(OXA) prepared in this invention uses natural soybean lecithin as a carrier and oxaloacetate (OXA) as a drug loading agent. It effectively overcomes the inherent defects of poor physicochemical properties and instability of natural lecithin-based nanomaterials, and solves the technical pain points of OXA's high polarity, weak cell membrane penetration, and low in vivo bioavailability. At the same time, it achieves the synergistic effect of the anticancer effect of soybean lecithin itself and the lactic acid inhibition effect of OXA. The material can specifically reduce the lactic acid concentration in tumor tissue and reverse the DC-T cell immunosuppression in the residual cancer microenvironment, and has excellent dual properties of tumor-targeted immunomodulation and anticancer.

[0027] This invention uses natural soybean lecithin as the core raw material. By optimizing the preparation process, it solves the technical problem of the difficulty in preparing high-performance nanomaterials from natural lecithin due to its complex composition, and successfully achieves efficient encapsulation and stable delivery of OXA. This preparation method differs from existing nanomaterial preparation methods using synthetic phospholipids (DPPA, DPPC, DSPC, etc.) as raw materials. It retains the high biocompatibility of natural lecithin, while also having the advantages of low raw material cost and strong process adaptability. It avoids the cost and biosafety shortcomings of synthetic phospholipid preparation processes, providing an optimized solution for the application of natural lecithin in nanomedicine delivery systems. Moreover, the physicochemical properties of the prepared nanomaterials are controllable, making it easier to achieve industrial scale-up.

[0028] This invention applies LEC (OXA) nanomaterials to the targeted therapy of residual cancer after IRFA (iron-radioactive arterial fat grafting) in mouse hepatocellular carcinoma orthotopic tumors. It significantly reduces lactate concentration in the residual cancer tissue, effectively reversing the lactate-mediated DC-T cell immunosuppressive microenvironment, thus achieving precise intervention in residual cancer after IRFA. The combined use of this material with PD-1 antibodies produces a significant synergistic therapeutic effect, achieving optimal efficacy in the treatment of residual cancer after IRFA. This provides a novel drug carrier and treatment strategy for the clinical combined immunotherapy of residual cancer after RFA in hepatocellular carcinoma. Furthermore, based on the application research of this material, the specific molecular mechanism by which increased lactate mediates DC-T cell immunosuppression in residual cancer after IRFA is elucidated. Inhibition of lactate production is identified as a key target for reversing immune resistance in residual cancer, providing important theoretical support for immunotherapy research on residual cancer after RFA. It also provides a universal therapeutic target and drug development strategy for tumor types with lactate-mediated immunosuppression, demonstrating broad application prospects in tumor immunotherapy. Attached Figure Description

[0029] Figure 1 The results of detection of key enzymes in cellular glycolysis in different treatment groups; among them, Figure 1 In this context, A represents the changes in the expression of key glycolytic enzymes in Hepa1-6 cells after sublethal thermal injury and hypoxia, analyzed by Western blotting (WB) experiments. Figure 1 In the figure, B represents a quantitative statistical graph of key enzymes in glycolysis.

[0030] Figure 2 To analyze the lactate concentration in the culture medium of Hepa1-6 cells after sublethal thermal injury and hypoxia.

[0031] Figure 3 The statistical results of pro-inflammatory factor expression levels after LPS-induced BMDC cells were treated with exogenous lactate are shown in the figure. Figure 3 In the figure, A represents the statistical results of Ifnb expression levels in cells of different treatment groups; Figure 3 In the figure, B represents the statistical results of Cxcl10 expression level; Figure 3 In the graph, C represents the statistical results of the expression level of Il-12b; Figure 3 In the figure, D represents the statistical results of Tnf-a expression level.

[0032] Figure 4 A statistical graph showing the results of flow cytometry analysis of the expression of surface co-stimulatory molecules and MHC class II molecules in LPS-induced BMDCs; among which, Figure 4 The A in the text represents CD80. + CD11c + Statistical results of the proportion of DC cells; Figure 4 The B in the text represents CD86. + CD11c+ Statistical results of the proportion of DC cells; Figure 4 In this context, C represents IA / IE. + CD11c + Statistical results of the proportion of DC cells.

[0033] Figure 5 The image shows the detection results of cGAS-STING pathway proteins in tumor tissues from different treatment groups; among them... Figure 5 A in the figure represents the Western blot electrophoresis diagram of cGAS-STING pathway protein levels before and after incomplete radiofrequency ablation of subcutaneous hepatocellular carcinoma in mice. Figure 5 B in the figure represents the quantitative statistical graph of the relative expression levels of key proteins in the cGAS-STING pathway.

[0034] Figure 6 Results of detection of key enzymes in glycolysis in tumor tissues from different treatment groups; among them... Figure 6 In the image, A represents the Western blot electrophoresis image of key glycolytic enzymes before and after radiofrequency ablation of incomplete subcutaneous hepatocellular carcinoma tumors in mice. Figure 6 The B in the figure represents a quantitative statistical graph showing the relative expression levels of key glycolytic enzymes before and after radiofrequency ablation of incomplete subcutaneous tumors in mouse liver cancer.

[0035] Figure 7 This figure shows the statistical results of ISGs mRNA expression levels in mouse subcutaneous hepatocellular carcinoma before and after incomplete radiofrequency ablation.

[0036] Figure 8 Flow cytometry images of surface molecular expression in dendritic cells (DCs) at different time points before and after radiofrequency ablation of incomplete subcutaneous hepatocellular carcinoma in mice.

[0037] Figure 9 CD80 at different time points before and after incomplete radiofrequency ablation of subcutaneous hepatocellular carcinoma in mice + CD11c + DC, CD86 + CD11c + DC, IA / IE + CD11c + A quantitative statistical chart of DC levels; among which... Figure 9 The A in the text represents CD80. + CD11c + Quantitative statistical results of the proportion of dendritic cells (DCs) at different time points; Figure 9 The B in the text represents CD86. + CD11c + Quantitative statistical results of the proportion of dendritic cells (DCs) at different time points; Figure 9 In this context, C represents IA / IE. + CD11c+ A graph showing the quantitative statistical results of the proportion of dendritic cells (DCs) at different time points.

[0038] Figure 10 Granzyme B at different time points before and after incomplete radiofrequency ablation of subcutaneous hepatocellular carcinoma in mice + CD8 + T cells, IFN-γ + CD8 + Image showing the results of flow cytometry analysis of T cells.

[0039] Figure 11 Granzyme B at different time points or in different treatment groups + CD8 + T cells, IFN-γ + CD8 + Quantitative statistical results of T cell proportion; among which... Figure 11 In this context, A represents different time points in CD8. + IFN-γ in T cells + Quantitative statistical results of cell proportions; Figure 11 In this context, B represents different time points CD8 + Granzyme B in T cells + Quantitative statistical results of cell proportions.

[0040] Figure 12 The graph shows the detection results of lactate concentration in tumor tissues of different treatment groups.

[0041] Figure 13 The figure shows the basic characterization and detection results of lecithin nanomaterials loaded with oxalate; among them, Figure 13 In this diagram, A represents the structural schematic of LEC (OXA); Figure 13 In the diagram, B represents the particle size distribution of LEC (OXA); Figure 13 In the diagram, C represents the potential diagram of LEC (OXA); Figure 13 In the image, D represents the electron microscope image of LEC (OXA).

[0042] Figure 14 The results are for drug release and stability testing of LEC (OXA); among them, Figure 14 In the diagram, A represents the cumulative drug release of LEC (OXA) at room temperature and 37°C. Figure 14 The B in the figure represents the particle size stability test results of LEC (OXA) at room temperature and 37°C.

[0043] Figure 15 This is a graph showing the results of cell uptake detection; where, Figure 15In the figure, A represents the uptake of Cy5 and LEC(Cy5) in Hepa1-6 cells at different time points; Figure 15 The B in the figure represents a statistical graph of Cy5 and LEC(Cy5) uptake at different time points in Hepa1-6 cells.

[0044] Figure 16 The graph shows the results of pharmacokinetics and tumor enrichment detection in mice; among them... Figure 16 In the diagram, A represents the pharmacokinetic diagram of LEC(Cy5) in mice over 24 hours. Figure 16 In the figure, B represents the drug distribution of Cy5 and LEC (Cy5) in major organs and tumors in a mouse subcutaneous hepatocellular carcinoma model; Figure 16 The figure 'C' represents a quantitative statistical analysis of the distribution of Cy5 and LEC (Cy5) in major organs and tumors in a mouse subcutaneous hepatocellular carcinoma model.

[0045] Figure 17 This is a graph showing the results of in vivo toxicity testing; among them, Figure 17 In the image, A represents the H&E staining of the major organs of mice after different treatments. Figure 17 In the figure, B represents the statistical results of serum ALT in mice after different treatments; Figure 17 In the figure, C represents the statistical results of serum AST in mice after different treatments; Figure 17 The figure shows the statistical results of serum BUN in mice after different treatments. Figure 17 The figure shows the statistical results of Crea levels in the serum of mice after different treatments.

[0046] Figure 18 The graph shows the results of detecting lactate production, proliferation, and colony formation in cells of different treatment groups; among them, Figure 18 The figure shows the statistical results of how different concentrations of Oxamate inhibited lactate production in Hepa1-6 cells after sublethal heat injury and hypoxia. Figure 18 The figure B represents the statistical results of the inhibition of lactate production in Hepa1-6 cells after treatment with multiple concentrations of LEC (OXA) following sublethal thermal injury and hypoxia. Figure 18 In the figure, C represents the CCK8 experimental results of IRFA cells after different culture medium treatments; Figure 18 In the diagram, D represents the staining image of IRFA cells in a plate colony assay after different culture medium treatments; Figure 18 In the figure, E represents the quantitative statistical graph of IRFA cell plate cloning experiment after different culture medium treatments.

[0047] Figure 19 This image shows the mature phenotype and pro-inflammatory factor mRNA expression results of mouse bone marrow-derived dendritic cells (BMDCs); among them, Figure 19 In this context, A represents the flow cytometry detection of CD80. + CD11c + DC ratio chart; Figure 19 In this context, B indicates flow cytometry detection of CD86. + CD11c + DC ratio chart; Figure 19 In this context, C indicates flow cytometry detection of IA / IE. + CD11c + DC scale chart; Figure 19 In the diagram, D represents the relative expression level of the pro-inflammatory factor Ifnb mRNA; Figure 19 In the diagram, E represents the relative expression level of the pro-inflammatory factor Cxcl10 mRNA; Figure 19 In the diagram, F represents the relative expression level of the pro-inflammatory factor Tnf-a mRNA; Figure 19 In the graph, G represents the relative expression level of pro-inflammatory factor Il-12b mRNA.

[0048] Figure 20 For mouse CD8 + Figure showing the expression results of functional molecules after T cells were co-cultured with BMDCs under different treatment conditions.

[0049] Figure 21 CD8 + IFN-γ in T cells + Cell ratio and Granzyme B + A quantitative statistical graph of cell proportions; among which, Figure 21 The A in the text represents CD8 + IFN-γ in T cells + Statistical results of cell proportions; Figure 21 In this context, B represents CD8. + Granzyme B in T cells + Statistical results of cell proportions.

[0050] Figure 22 This is a schematic diagram of the treatment model for an IRFA model of orthotopic hepatocellular carcinoma in mice.

[0051] Figure 23 Ultrasound images of mice at different time points after treatment with PBS, Oxamate, LEC, LEC (OXA), and LEC (OXA) + anti-PD-1 antibody; the green circle represents the cross-section of the tumor.

[0052] Figure 24 The figures show the tumor volume changes and mouse body weight changes in mice after treatment with PBS, Oxamate, LEC, LEC (OXA), and LEC (OXA) + anti-PD-1. Figure 24In this context, A represents the tumor volume change curves in mice under different treatment groups; Figure 24 In the figure, B represents the curve of change in body weight of mice in different treatment groups.

[0053] Figure 25 The lactate content in tumor tissue of different treatment groups in a mouse hepatocellular carcinoma orthotopic tumor IRFA model.

[0054] Figure 26 For the detection of CD80 in tumor tissue by flow cytometry + CD11c + DC, CD86 + CD11c + DC, IA / IE + CD11c + A plot of representative scatter points of DC.

[0055] Figure 27 A quantitative statistical graph of the mature phenotype of dendritic cells (DCs) in tumor tissue; among which, Figure 27 In this context, A represents CD11c. + CD80 in cells + Statistical results of the proportion of DC; Figure 27 In this context, B represents CD11c. + CD86 in cells + Statistical results of the proportion of DC; Figure 27 The C in the text represents CD11c. + IA / IE in cells + A statistical chart showing the proportion of DC (Distributed Capacity).

[0056] Figure 28 After different treatment regimens, CD8 levels in the tumor tissue of a mouse hepatocellular carcinoma orthotopic tumor IRFA model were observed. + Figure showing the results of T cell infiltration and functional molecule expression.

[0057] Figure 29 After different treatment regimens, CD8 levels in the tumor tissue of a mouse hepatocellular carcinoma orthotopic tumor IRFA model were observed. + The results of quantitative statistical analysis of T cell infiltration and functional molecule expression are shown in the figure; among them... Figure 29 The A in the text represents CD8 + A graph showing the statistical proportion of T cell infiltration in tumor tissue; Figure 29 In this context, B represents IFN-γ + CD8 + T cells in CD8 + A graph showing the proportion of T cells; Figure 29 In this context, C represents Granzyme B. + CD8 + T cells in CD8 +A graph showing the proportion of T cells. Detailed Implementation

[0058] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0059] The following description is based on specific embodiments.

[0060] Example 1: Effects of lactate on DC maturation and CD8 after IRFA surgery + The impact of T cell function I. Cellular level (a) Experimental cells: Mouse liver cancer Hepa1-6 cells, purchased from Wuhan Pronosai Life Science Technology Co., Ltd.; mouse primary bone marrow-derived dendritic cells (BMDC), used for in vitro lactate intervention experiments.

[0061] Construction of primary mouse BMDC cells (extraction and culture): Male C57BL / 6 mice aged 6–8 weeks were used. The hind limb femurs of both sides were isolated, and the skin and muscle tissue on the surface of the femurs were removed to expose the intact femurs. The bone marrow cavity was flushed with DMEM high-glucose medium, and the bone marrow suspension was collected. The suspension was filtered through a 70 μm cell filter to remove impurities. The filtered bone marrow suspension was centrifuged to obtain mouse primary bone marrow cells. Mouse primary BMDC cells were screened by flow cytometry and seeded in RMI 1640 medium containing GM-CSF at a final concentration of 20 ng / ml. The cells were cultured routinely for subsequent exogenous lactate intervention experiments.

[0062] (II) Cell Model Construction 1. Heat injury group: Hepa1-6 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin by volume. When the cell confluence reached about 70%~80%, the cells were heated in a water bath at 45°C for 10 min to induce sublethal heat injury and form a heat injury model.

[0063] 2. Hypoxia model: Hepa1-6 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin by volume. When the cell confluence reached about 70%~80%, they were placed in a 94%N2+5%CO2+1%O2 cell culture incubator and cultured for 48 hours to form a hypoxia model.

[0064] 3. In vitro IRFA cell model (Heat+Hypoxia): Hepa1-6 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. When the cell confluence reached about 70%~80%, the cells were heated in a 45°C water bath for 10 min to cause sublethal heat damage. Then, they were cultured in a 94% N2+5% CO2+1% O2 cell culture incubator for 48 h to obtain the in vitro IRFA cell model.

[0065] 4. Control group (Ctrl): Hepa1-6 cells were not subjected to heat damage and hypoxia treatment, and were kept in a conventional incubator (37℃, 5% CO2) and cultured synchronously with other groups until the corresponding time points were collected.

[0066] (III) In vitro lactate intervention experiment Primary mouse BMDC cells were extracted using the method described above. 100 ng / mL LPS was added to the primary BMDC cells cultured for 7 days to induce cell activation. Simultaneously, lactate at final concentrations of 5 mM and 20 mM was added to the activated BMDC cells for intervention. The cells were then placed in an incubator and cultured for another 6 hours. Relevant indicators were then measured after the culture was completed.

[0067] (iv) Testing methods 1. Following the above method for constructing cell models, different cell models were constructed. After successful construction, total RNA was extracted from each group of cells, reverse transcribed into cDNA, and then the expression levels of key glycolytic enzymes (PKM2 and LDHA) in different group of model cells were detected by real-time quantitative PCR.

[0068] 2. Lactic acid content determination: Take the supernatant of cell culture medium from each group after centrifugation, and use a lactic acid detection kit (enzyme-catalyzed colorimetric method) to measure the absorbance at the corresponding wavelength using an ELISA reader, strictly following the instructions of the kit. Calculate and compare the lactic acid content in each group of tumor tissues based on the standard curve.

[0069] 3. In an in vitro lactate intervention experiment, total RNA was extracted from LPS-induced bone marrow-derived dendritic cells (BMDCs) treated with different concentrations of exogenous lactate. After reverse transcription into cDNA, the expression levels of pro-inflammatory factors Ifnb, Cxcl10, Il-12b, and Tnf-a mRNA were detected using real-time quantitative PCR. CD80 was analyzed by flow cytometry. + CD11c + DC, CD86 + CD11c + DC, IA / IE + CD11c + The proportion of DC double-positive cells is used to determine the maturation and activation level of BMDCs.

[0070] (V) Experimental Results 1. Results of expression levels of key glycolytic enzymes (PKM2, LDHA) in various cell models like Figure 1 As shown in A and B, sublethal heat injury and hypoxia treatment significantly increased the expression levels of key glycolytic enzymes in Hepa1-6 cells.

[0071] 2. Results of lactate secretion detection in the culture media of each cell model like Figure 2 As shown, sublethal heat injury and hypoxia treatment significantly increased the lactate concentration in the culture medium of Hepa1-6 cells, suggesting that sublethal heat injury and hypoxia can induce increased lactate secretion in Hepa1-6 cells.

[0072] 3. Detection of pro-inflammatory factor mRNA in BMDC cells like Figure 3 As shown in Figures A through D, after exogenous lactate treatment of LPS-induced BMDC cells, the mRNA expression levels of pro-inflammatory factors Ifnb, Cxcl10, Il-12b, and Tnf-a were significantly reduced, indicating that exogenous lactate can inhibit the secretion of pro-inflammatory factors in BMDC cells.

[0073] 4. BMDC cell maturation and activation detection like Figure 4 As shown in A~C, after exogenous lactate treatment of LPS-induced BMDC cells, CD80 + CD11c + DC, CD86 + CD11c + DC, IA / IE + CD11c + The DC level was significantly reduced, confirming that exogenous lactic acid can significantly inhibit LPS-induced BMDC maturation and activation.

[0074] II. Animal Experiments (I) Laboratory Animals and Cells 1. Experimental animals: SPF-grade C57BL / 6 male mice, divided into two age groups: 3-5 weeks old (weight 15-18 g), used to construct a subcutaneous hepatocellular carcinoma model; 6-8 weeks old, used to extract primary BMDC cells. All mice were housed in an SPF-grade environment.

[0075] 2. Experimental cells: Mouse liver cancer Hepa1-6 cells, purchased from Wuhan Pronosai Life Science Technology Co., Ltd.

[0076] (II) Mouse Model Construction 1. Construction of subcutaneous hepatocellular carcinoma tumors in mice: 3-5 week old male C57BL / 6 mice weighing 15-18 g were subcutaneously injected into both backs with 100 μL of Hepa1-6 cell suspension containing 50% matrix gel (containing 2×10^6 cells). The mice were then fed according to standard feeding procedures until the tumor volume grew to approximately 500 mm. 3 At that time, the model was completed and will be used for subsequent IRFA treatment and related testing.

[0077] 2. IRFA treatment of subcutaneous hepatocellular carcinoma tumors in mice: When the tumor volume in the subcutaneous tumor model reaches 500 mm... 3 At that time, the left tumor of the mouse was removed and used as the control group (Ctrl group) before IRFA treatment; the right tumor of the mouse was treated with incomplete radiofrequency ablation (IRFA) and recorded as the IRFA group; the right tumor tissue of the mouse was collected on the 3rd, 7th and 10th days after IRFA treatment (D3, D7 and D10); 3 biological replicates were set up for each of the Ctrl group and the IRFA group, and the treatment and detection were carried out simultaneously.

[0078] (III) Detection Methods 1. Western blot detection (1) Total protein was extracted from tumor tissues at each time point in the Ctrl group and IRFA group. Western blot was used to detect the expression and phosphorylation levels of key proteins (cGAS, STING, p-STING, TBK1, p-TBK1, IRF3, p-IRF3) in the cGAS-STING signaling pathway. β-actin was used as an internal reference protein to correct for differences in loading amount.

[0079] (2) Detection of key enzymes in glycolysis: Tumor tissues from each group were collected, total protein was extracted, and the expression levels of PKM2 and LDHA proteins were detected by Western blot.

[0080] 2. qRT-PCR detection Total RNA was extracted from tumor tissues at each time point in the Ctrl and IRFA groups, reverse transcribed into cDNA, and the mRNA expression level of interferon-stimulated genes (ISGs) was detected by real-time quantitative PCR.

[0081] 3. Flow cytometry analysis Tumor tissues from the Ctrl and IRFA groups at various time points were enzymatically digested to prepare single-cell suspensions. Flow cytometry was used to detect the expression levels of CD80, CD86, and IA / IE on the surface of dendritic cells (DCs) within the tumor, as well as CD8+. + Expression levels of GranzymeB and IFN-γ in T cells.

[0082] 4. Lactic acid content determination Tumor tissues from the Ctrl and IRFA groups at various time points were collected, and tissue homogenates were prepared on ice. After centrifugation, the supernatant was collected. The absorbance at the corresponding wavelength was measured using an enzyme-linked immunosorbent assay (ELISA) kit, strictly following the kit instructions. The lactate content in the tumor tissues of each group was calculated and compared based on the standard curve.

[0083] (iv) Experimental Results 1. Western blot results (1) Detection of cGAS-STING pathway proteins in tumor tissue like Figure 5 As shown in A and B, after IRFA surgery, the phosphorylation levels (p-STING, p-TBK1, p-IRF3) of key proteins (cGAS, STING, TBK1, IRF3) in the cGAS-STING signaling pathway in residual cancer cells were significantly increased, while the total protein expression did not change significantly, confirming that IRFA can activate the cGAS-STING pathway in residual cancer cells.

[0084] (2) Detection of key enzymes in tumor tissue glycolysis like Figure 6 In cases A and B, after IRFA surgery, the expression levels of key glycolytic enzymes (PKM2 and LDHA) in residual cancer tissue were significantly upregulated.

[0085] 2. qRT-PCR detection results Results of ISGs mRNA detection in tumor tissue as follows Figure 7 As shown, after IRFA surgery, the mRNA expression level of interferon-stimulated genes (ISGs) in tumor tissue was significantly upregulated, consistent with the results of cGAS-STING pathway activation.

[0086] 3. Flow cytometry analysis results (1) Detection of DC cells in tumor tissue: such as Figure 8 and Figure 9 As shown in A~C, the intratumoral CD80 levels at different time points (D3, D7, D10) after IRFA surgery. + CD11c + DC, CD86 + CD11c + DC, IA / IE + CD11c + The DC levels were significantly reduced, suggesting that the maturation and activation of DC cells within the tumor were significantly suppressed after IRFA surgery.

[0087] (2) CD8 in tumor tissue + T-cell detection: such as Figure 10 and Figure 11As shown in A and B, Granzyme B in the tumor at different time points after IRFA surgery + CD8 + T cells, IFN-γ + CD8 + The levels of T cells were significantly reduced, indicating that the internal CD8+ cells in the tumor were significantly reduced after IRFA surgery. + T cell function was significantly suppressed.

[0088] 4. Results of lactic acid content determination Results of lactate concentration detection in tumor tissue as follows Figure 12 As shown, after IRFA surgery, the concentration of lactic acid in the residual cancer tissue increased significantly.

[0089] In summary, this indicates that increased lactate in the residual cancer microenvironment after IRFA surgery mediates DC-CD8. + The molecular mechanism of T-cell immunosuppression was investigated, and lactate was identified as a key target for reversing residual cancer immune resistance, providing core theoretical support for the design of subsequent treatment plans.

[0090] Example 2: Lecithin nanomaterials loaded with oxalate and their preparation method (I) A method for preparing lecithin nanomaterials loaded with oxamate (LEC(OXA)): specifically including the following steps: Soy lecithin (LEC) was dissolved in chloroform to form an LEC / chloroform solution with a concentration of 10 mg / ml; DSPE-PEG2000 was dissolved in methanol to form a DSPE-PEG2000 / methanol solution with a concentration of 10 mg / ml.

[0091] Take 200 μL of the prepared LEC / chloroform solution and 20 μL of DSPE-PEG2000 / methanol solution, mix them, and the mass ratio of lecithin to DSPE-PEG2000 is 10:1. Transfer the mixture to a brown glass bottle and evaporate it to dryness in a fume hood to form a uniform film on the inner wall of the glass bottle.

[0092] Oxamate (manufacturer: MCE; product number: HY-W013032A) was dissolved in pure water to form a 62.5 mg / ml Oxamate solution. 1 ml of the Oxamate solution was added to the brown bottle that had been evaporated to dryness. The mixture was magnetically stirred for 1 h (1200 rpm / min) to form an emulsion. The emulsion was sonicated at 160 W for 20 s. After centrifugation through an ultrafiltration tube and purification by washing with PBS, the supernatant was LEC (OXA).

[0093] (II) Detection Indicators and Detection Methods 1. Basic characterization and detection of nanomaterials For the nanomaterials prepared by the above method, particle size, potential, morphology, drug release, and stability were measured to clarify the basic properties of the materials. The specific testing methods are as follows: Particle size, potential and morphology detection: The particle size and potential of LEC (OXA) were detected by dynamic light scattering instrument; the microstructure of LEC (OXA) was observed by 120kV transmission electron microscopy, and relevant characterization data were recorded.

[0094] Drug release level and stability assay: LEC (Cy5) samples and an equal volume of pure Cy5 solution were placed in dialysis tubes with a molecular weight cutoff of 100 kDa. The dialysis tubes were immersed in PBS buffer and incubated at room temperature and 37°C (simulating the in vivo environment), respectively. Samples were taken at 0.5, 1, 2, 4, 6, 8, 12, and 24 h to detect the fluorescence intensity of Cy5 in the samples. The drug release level of the nanomaterials (nanoliposomes) was determined based on the changes in fluorescence intensity. At the same time, the particle size of the nanomaterials was detected at the above time points, and the stability under different environments and incubation times was evaluated by the trend of particle size change.

[0095] 2. Detection of cellular uptake of nanomaterials Hepa1-6 tumor cells were seeded in six-well plates and cultured to a suitable density (cell confluence approximately 80%). Then, 2 ml of Cy5 solution and an equal volume of LEC (Cy5) were added to each well. Cells were collected at 0.5, 1, 2, and 4 h, and the fluorescence level of Cy5 in the cells was detected. By analyzing the differences and changes in intracellular fluorescence intensity, the uptake efficiency and uptake kinetics of nanomaterials (LEC (Cy5)) and pure Cy5 by Hepa1-6 cells were analyzed.

[0096] 3. In vivo tumor enrichment detection of nanomaterials Six male C57BL / 6 mice aged 3-5 weeks and weighing 15-18g were selected. After preparing and disinfecting the backs of the mice, 1 million Hepa1-6 tumor cells were percutaneously injected using a 1ml syringe to construct a subcutaneous tumor model in mice. The tumors were allowed to grow to 200mm in size. 3 Mice were randomly divided into two groups of three (n=3) each: a control group (injected with 200 μl of free Cy5 solution at a concentration of 10 μM) and an experimental group (injected with 200 μl of LEC(Cy5) at the same concentration as the control group). The corresponding samples were injected into the mice in both groups via the tail vein. After 24 hours of culture, the mice were sacrificed, and their heart, liver, spleen, lung, kidney, tumor tissue, and muscle tissue were dissected and collected. The fluorescence intensity of Cy5 in each tissue was detected, and the fluorescence distribution and intensity in each tissue, especially the tumor tissue, of the two groups of mice were compared to analyze the in vivo tumor enrichment ability of the nanomaterials.

[0097] 4. In vivo toxicity testing of nanomaterials The specific methods for detecting the toxicity of nanomaterials in animals and assessing their biosafety are as follows: Thirty-five male C57BL / 6 mice aged 6-8 weeks and weighing 19-22g were randomly divided into 5 groups of 7 mice each (n=7): blank control group (PBS group), Oxamate control group (Free OXA), LEC control group, LEC(OXA) experimental group, and LEC(OXA) + Anti-PD-1 antibody combined experimental group (LEC(OXA) + Anti-PD-1). All mice were administered the drugs via tail vein injection for 3 consecutive days. The dosages were as follows: blank control group received PBS; free OXA control group received OXA 300 mg / kg; LEC control group received LEC 32 mg / kg; LEC(OXA) experimental group received LEC(OXA) drug-loaded formulation (OXA loading rate 30%), which, based on an absolute OXA dose of 300 mg / kg, was equivalent to a dosage of 332 mg / kg; LEC(OXA) + Anti-PD-1 group received LEC(OXA) formulation 332 mg / kg (containing OXA). 300 mg / kg, LEC 32 mg / kg), combined with 100 μg / animal anti-PD-1 antibody.

[0098] After administration, serum and tissues from the heart, liver, spleen, lung, and kidneys of mice were collected. Serum levels of liver and kidney function-related indicators (such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Crea), blood urea nitrogen (BUN), etc.) and inflammatory factors were detected. Pathological morphological changes in various organs and tissues were observed, and the in vivo toxicity of nanomaterials (and combined drugs) was comprehensively evaluated.

[0099] (III) Experimental Results: 1. Basic characterization test results like Figure 13 As shown in Figures A through D, lecithin nanomaterials LEC (OXA) loaded with Oxamate were successfully constructed, with a particle size of 206.1 ± 8.9 nm and an electrical potential of -10.8 ± 0.9 mV.

[0100] like Figure 14 As shown in A and B, the constructed nanomaterials exhibit good slow drug release capability in PBS at room temperature and 37°C, as well as good stability in PBS or PBS+10% FBS at room temperature and 37°C.

[0101] 2. Cell uptake detection results like Figure 15As shown in Figures A and B, at different time points, Hepa1-6 cells took up LEC (Cy5) better than free Cy5. This demonstrates the good cellular uptake capability of the constructed nanomaterials.

[0102] 3. In vivo pharmacokinetics and tumor enrichment detection results like Figure 16 As shown in A~C, LEC (Cy5) has a longer circulation time in vivo compared to free Cy5 and has a good tumor enrichment capacity, which can significantly improve the efficiency of targeted drug delivery in vivo.

[0103] 4. In vivo toxicity test results like Figure 17 As shown in A~E, in vivo studies have demonstrated that Oxamate, LEC, LEC(OXA), and LEC(OXA) + anti-PD-1 antibody have no significant toxic side effects on the heart, liver, spleen, lungs, and kidneys.

[0104] Example 3: Verification of the effects of Oxamate-loaded lecithin nanomaterials at the cellular level (I) Validation of the effect of tumor cell metabolic intervention This study aims to verify whether free Oxamate and drug-loaded nanoparticles LEC (OXA) can inhibit lactate production, proliferation activity, and colony formation in IRFA model tumor cells, clarify their intervention effects on tumor cell glycolysis and malignant proliferation, identify the core metabolic intervention role, and lay the foundation for subsequent research on immune regulation mechanisms.

[0105] 1. The Hepa1-6 mouse hepatocellular carcinoma cell line was purchased from Wuhan Pronosai Life Science Technology Co., Ltd., and stored in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0106] 2. Construction of IRFA cell model IRFA cell model construction: Hepa1-6 cells were cultured until the cell confluence reached about 70%~80%, and then heated in a 45℃ water bath for 10 min to cause sublethal heat damage. They were then placed in a 94%N2+5%CO2+1%O2 cell culture incubator and cultured for 48 h to form an IRFA cell model.

[0107] 3. Grouping (1) Control group (Ctrl): Hepa1-6 cells cultured until the cell confluence reached about 70%~80%; (2) IRFA cell model (Heat+Hypoxia): The constructed IRFA cell model was placed in a complete culture medium and cultured in a 95% air + 5% CO2 cell culture incubator for 48h.

[0108] (3) Free Oxamate group (OXA): The constructed IRFA cell model was placed in a complete culture medium with a final concentration of 10mM, 20mM, 30mM, 40mM and 50mM of free Oxamate and cultured in a 94%N2+5%CO2+1%O2 cell culture incubator for 48h.

[0109] (4) LEC(OXA) group: The constructed IRFA cell model was placed in LEC(OXA) complete medium containing 10 mM, 20 mM, 30 mM, 40 mM and 50 mM Oxamate, and cultured in a 94% N2 + 5% CO2 + 1% O2 cell culture incubator for 48 h.

[0110] The complete culture medium used in the above groups was based on DMEM medium with the addition of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0111] 4. Detection Indicators and Methods Lactic acid content: Cell culture supernatant from each group (including free Oxamate, LEC (OXA) gradient concentration groups and all control groups) was collected. The lactate detection kit was used with an enzyme-catalyzed colorimetric method. The kit was operated strictly according to the instructions. The absorbance value at the corresponding wavelength was measured with an ELISA reader. The lactate production of each group was calculated according to the standard curve. At the same time, the intervention effect at 30mM concentration was verified.

[0112] Cell proliferation activity: The CCK8 assay was used to detect the proliferation activity of cells cultured to a set time point. The effects of different treatments on the short-term proliferation of tumor cells were analyzed. The groups with a final Oxamate concentration of 30 mM (control group, IRFA cell model group, free Oxamate 30 mM group, LEC (OXA) 30 mM group) were selected from the above groups.

[0113] Colony formation ability: A plate colony formation assay was used. Groups with a final Oxamate concentration of 30 mM (control group, IRFA cell model group, free Oxamate 30 mM group, LEC (OXA) 30 mM group) were selected from the above groups. After culture, the cells were fixed and stained, and the number of cell colonies formed in each group was counted to evaluate the effect of different treatments on the long-term colony formation ability of tumor cells.

[0114] 5. Test Results The results are as follows Figure 18As shown in Figures A through E, Oxamate and LEC (OXA) can significantly inhibit lactate production in IRFA tumor cells, while also inhibiting cell proliferation and colony formation. Furthermore, the intervention effect of LEC (OXA) is superior to that of free Oxamate, confirming that this nano-drug delivery system can effectively target and inhibit glycolytic metabolism in tumor cells.

[0115] (II) Verification of tumor immune regulation mechanism This part of the experiment focuses on "lactic acid accumulation in the hypoxic tumor microenvironment - inhibition of BMDC maturation and activation - blocking CD8". + The core mechanism of "T cell anti-tumor immunity" was explored. Through progressive group verification, the inhibitory effect of IRFA tumor supernatant on immune cells was clarified, as well as the reversal effect of Oxamate and LEC (OXA) on the immunosuppressive microenvironment by regulating glycolysis and reducing lactate production.

[0116] 1. Preparation of experimental cells Hepa1-6 mouse hepatocellular carcinoma cell line was purchased from Wuhan Pronosei Biotechnology Co., Ltd., and stored in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0117] Sublethal thermal damage Hepa1-6 cells were cultured in complete culture medium until the cell confluence reached about 70%~80%. Then, they were heated in a water bath at 45℃ for 10 min to induce sublethal thermal damage. After that, they were placed in a normal oxygen (95% air + 5% CO2) environment and cultured for 48 h for later use.

[0118] Sublethal thermal damage Hepa1-6 cell hypoxia culture: Hepa1-6 cells were cultured in complete culture medium until the cell confluence reached about 70%~80%. Sublethal thermal damage was caused by heating in a 45℃ water bath for 10 min. Then, the cells were placed in a hypoxic environment (94% N2 + 5% CO2 + 1% O2) and cultured for 48 h for later use.

[0119] Mouse primary BMDC cell extraction and culture: The extraction method is the same as in Example 1. The cells are induced and cultured in vitro until day 7 for later use in maturation and activation detection and T cell co-culture.

[0120] mouse primary CD8 + T-cell extraction and activation: Mouse spleens were harvested, ground, and passed through a 40 μm filter. After centrifugation at 400 g / min for 8 min, red blood cells were removed using erythrocyte lysis buffer. The cells were washed once with PBS and centrifuged again at 400 g / min for 8 min. CD8+ cells were then separated using magnetic beads. + T cells were co-cultured with induced activated BMDCs in 96-well plates for 48 hours before use.

[0121] 2. Culture medium The complete culture medium is based on DMEM medium, with the addition of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0122] Normative tumor culture medium: It consists of 60% by volume of culture medium containing sublethal heat-damaged Hepa1-6 cells cultured in normoxic conditions for 48 hours and 40% by volume of fresh complete culture medium.

[0123] Hypoxia medium: It consists of 60% (v / v) of medium containing sublethal heat-damaged Hepa1-6 cells cultured under hypoxia for 48 h and 40% (v / v) of fresh complete medium.

[0124] Hypoxia + Oxamate tumor culture medium: It consists of a medium containing 60% by volume of sublethal heat-damaged Hepa1-6 cells cultured under hypoxia for 48 h with a final concentration of 30 mM free Oxamate, and 40% by volume of fresh complete culture medium.

[0125] Hypoxia + LEC (OXA) tumor culture medium: It consists of a medium containing 60% LEC (OXA) at a final concentration of 30mM, which has been used to culture sublethal heat-damaged Hepa1-6 cells under hypoxia for 48 hours, and 40% fresh complete culture medium.

[0126] 3. Experimental Methods Primary mouse BMDCs were induced and cultured in vitro until day 7. Maturation and activation were induced by adding 100 ng / ml LPS. Complete culture medium (labeled I), normoxic tumor medium (labeled II), hypoxic tumor medium (labeled III), hypoxic + Oxamate tumor medium (labeled IV), and hypoxic + LEC (OXA) tumor medium (labeled V) were added and cultured in an incubator for 6 hours to complete the intervention treatment. Flow cytometry was then used to detect the expression levels of CD80, CD86, and IA / IE, and RNA was extracted to detect the mRNA expression levels of pro-inflammatory factors (Ifnb, Cxcl10, Il-12b, Tnf-a).

[0127] After the intervention, BMDC and CD8 were collected from each of the above groups. + T cell co-culture system was constructed and incubated in 96-well cell culture plates for 48 hours, followed by CD8 detection. + Granzyme B and IFN-γ expression levels in T cells.

[0128] In the co-cultivation system, BMDC and CD8 +The T cell ratio was 10w:50w, and the culture medium was 200μL of 1640 complete medium per well. The culture conditions were 37℃, 5% CO2, and saturated humidity for 48 h.

[0129] The 1640 complete medium is based on RPMI 1640 medium with the addition of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0130] 4. Test Results (1) Results of CD80, CD86, and IA / IE expression level detection like Figure 19 As shown in A~C, this illustrates that the hypoxic medium inhibited BMDC activation, and the addition of Oxamate or LEC (OXA) could reverse this inhibitory effect.

[0131] (2) Results of pro-inflammatory factor detection like Figure 19 As shown in D~G, this indicates that hypoxic culture medium inhibits the secretion of the pro-inflammatory factor IL-12 by BMDCs, and the addition of Oxamate or LEC (OXA) can reverse this inhibitory effect.

[0132] (3) CD8 + Granzyme B and IFN-γ expression levels on T cells like Figure 20 and Figure 21 As shown in A and B, this demonstrates that Oxamate or LEC (OXA) inhibits lactate production and can reverse the suppression of BMDC-mediated T-cell immune function by hypoxic tumor supernatant.

[0133] In summary, this demonstrates that sublethal thermal injury and hypoxic Hepa1-6 tumor supernatant inhibit LPS-induced BMDC maturation and IL-12 synthesis, thereby inhibiting CD8. + T cell function was restored by both free Oxamate and drug-loaded nanoparticles LEC (OXA) effectively reversing the aforementioned inhibitory effects by inhibiting lactate production in tumor cells, thereby restoring CD8 function. + The anti-tumor immune function of T cells; at the cellular level, the inhibitory effects of free Oxamate and LEC (OXA) are comparable, suggesting that in vivo, drug-loaded nanoparticles LEC (OXA) will exhibit better intervention effects.

[0134] Example 4: Validation of the effects of Oxamate-loaded lecithin nanomaterials in animal models (a) Experimental materials 1. Laboratory animals Male C57BL / 6 mice aged 3-5 weeks and weighing 15-18g were purchased from the Laboratory Animal Center of Sun Yat-sen University. All in vivo experiments were conducted in accordance with the guidelines approved by the International Association for the Protection and Use of Animals (IACUC) of Sun Yat-sen University; Ethics No.: AP20250152.

[0135] 2. Establishment of a mouse model of hepatocellular carcinoma in situ (IRFA): 3-5 week old male C57BL / 6 mice weighing 15-18g were anesthetized, skin prepared, disinfected, and their livers exposed. 50 μl of Hepa1-6 cell suspension containing 50% matrix gel was injected via insulin injection, and the peritoneal cavity was closed. When the in situ tumor volume reached 50 mm³, radiofrequency ablation was performed under ultrasound guidance. The radiofrequency needle was inserted into one side of the tumor, and treatment was stopped after 3 weeks and 30 seconds. After hemostasis, the peritoneal cavity was closed. Simultaneously, the volume of residual tumor after hepatocellular carcinoma ablation was observed every three days using three-dimensional ultrasound scanning, and the mice's weight was recorded.

[0136] (II) Grouping and Administration: like Figure 22 As shown, mice with successfully constructed IRFA hepatocellular carcinoma models were selected and randomly divided into 5 groups of 5 mice each using a completely randomized method. All groups received the drug via tail vein injection starting from the second day after tumor ablation. The specific grouping, administration method, dosage, and frequency are as follows: PBS group (denoted as a): PBS intervention was administered alone; the dosage was 200 μL per mouse; The Oxamate group (OXA, denoted as b) was treated with Oxamate alone at a dose of 300 mg / kg (based on mouse body weight) every 2 days for 3 consecutive times. LEC group (denoted as c): LEC preparation was administered alone; the dosage was 32 mg / kg (calculated based on mouse body weight), and the frequency of administration was once every 2 days for 3 consecutive times; The LEC(OXA) group (denoted as d) was treated with the LEC(OXA) prepared in Example 2. Based on an absolute LEC(OXA) dose of 300 mg / kg (calculated per mouse body weight), and considering the 30% encapsulation rate of OXA in the LEC formulation, the actual dosage was calculated. The administration frequency was once every two days for three consecutive times. LEC(OXA) + anti-PD-1 antibody group (referred to as e): The same dose of LEC(OXA) combined with anti-PD-1 antibody intervention was given to the LEC(OXA) group; the dosage, method and frequency of LEC(OXA) administration were completely consistent with the LEC(OXA) group, and anti-PD-1 antibody was administered in combination at a dose of 100 μg / animal, via tail vein injection, once every 3 days.

[0137] (III) Testing methods: (1) Tumor growth determination: After the administration of the drug, the residual cancer foci of each group of mice were removed and their mass and volume were measured.

[0138] (2) Lactic acid level determination: After the administration was completed, the residual cancer lesions of each group of mice were removed, and a portion of the tissue was homogenized and the lactic acid level was measured. The specific determination method was the same as described in Example 1.

[0139] (3) Determination of immune activation in residual tumor cells: Residual tumor foci were removed from mice in each group, and half of the residual tumor cells were enzymatically digested into single-cell suspensions. CD80 levels were detected by flow cytometry. + CD11c + DC, CD86 + CD11c + DC, IA / IE + CD11c + DC level, CD8 + T cell infiltration and CD8 + Expression of Granzyme B and IFN-γ in T cells. The other half of the residual cancer cell was treated with 4% paraformaldehyde, embedded in paraffin, and sectioned.

[0140] (iv) Experimental Results (1) Tumor growth in mice like Figure 23 , Figure 24 As shown in A and B, in a mouse hepatocellular carcinoma orthotopic tumor (IRFA) model, the LEC(OXA) + anti-PD-1 antibody treatment group demonstrated that it achieved the strongest inhibition of residual tumor growth.

[0141] (2) Lactic acid concentration like Figure 25 As shown, in a mouse hepatocellular carcinoma orthotopic tumor IRFA model, it was demonstrated that LEC (OXA) can reduce the concentration of lactate in tumor tissue.

[0142] (3) Immunization results like Figure 26 and Figure 27 As shown in A~C, in the mouse hepatocellular carcinoma orthotopic tumor IRFA model, it was demonstrated that both LEC(OXA) and LEC(OXA) + anti-PD-1 treatment groups could enhance the maturation and activation of dendritic cells (DCs) in the tumor microenvironment.

[0143] like Figure 28 and Figure 29 As shown in A~C, in a mouse hepatocellular carcinoma orthotopic tumor IRFA model, it was demonstrated that LEC(OXA) and LEC(OXA) + anti-PD-1 treatment groups could maximally activate CD8. +T cell levels of Granzyme B and IFN-γ, and increased CD8 levels. + T cell infiltration level.

[0144] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0145] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A lecithin nanomaterial loaded with oxalate, characterized in that, The lecithin nanomaterial loaded with oxalate has a shell-core structure; the shell is a phospholipid bilayer structure formed by soybean lecithin and DSPE-PEG2000; and the core is oxalate.

2. The method for preparing lecithin nanomaterials loaded with oxalate salts according to claim 1, characterized in that, Includes the following steps: Soybean lecithin and DSPE-PEG2000 were dissolved separately in organic solvents, and then mixed to obtain a mixture; the mixture was rotary evaporated until a lipid film appeared. The oxalate salt is mixed with the lipid film, and the lipid film is hydrated to form an emulsion; The emulsion was purified by sonication, ultrafiltration centrifugation, and washing with PBS. The supernatant was lecithin nanomaterials loaded with oxalate.

3. The preparation method according to claim 2, characterized in that, The mass ratio of soybean lecithin, DSPE-PEG2000 and oxalate is 1.5~2.2:0.1~0.3:60~65.

4. The preparation method according to claim 2, characterized in that, The organic solvent includes one or both of chloroform and methanol.

5. The preparation method according to claim 2, characterized in that, The oxalate salt is mixed with the lipid film and stirred at 1000~1400 rpm / min for 0.8~1.2h to form an emulsion.

6. The preparation method according to claim 2, characterized in that, The power of the ultrasound on the emulsion is 150W~170W, and the duration is 18~23s.

7. The use of the oxaloacetate-loaded lecithin nanomaterial as described in claim 1 or the oxaloacetate-loaded lecithin nanomaterial prepared by any one of claims 2 to 6 in the preparation of a drug for treating residual tumors after radiofrequency ablation of cancer.

8. The application as described in claim 7, characterized in that, The cancers mentioned include liver cancer.

9. The application as described in claim 7, characterized in that, The drug comprises a combination of lecithin nanomaterials loaded with oxalate and a PD-1 antibody.

10. A drug for treating residual tumors after radiofrequency ablation for cancer, characterized in that, The lecithin nanomaterial loaded with oxalate as described in claim 1, and a pharmaceutically acceptable carrier.

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