Lactate oxidase nanocapsule and preparation method therefor

Abstract

A lactate oxidase nanocapsule and a preparation method therefor. The lactate oxidase nanocapsule is prepared by taking poly(ethylene glycol) methyl ether acrylate, 2-methacryloyloxyethyl phosphorylcholine, N-(3-aminopropyl)methacrylamide and / or acrylamide as monomers, taking N,N'-methylenebisacrylamide, glycerol dimethacrylate and / or triblock polylactic acid-polyethylene glycol-dimethacrylate as cross-linking agents, and using a free radical polymerization reaction to synthesize a nano-thickness polymer shell layer on the surface of lactate oxidase in situ. The lactate oxidase nanocapsule can eliminate lactic acid and release an immunostimulant, eliminating immunosuppression, can be used in combination with an immune checkpoint blocker, improving the immune curative effect of the immune checkpoint blocker.

Description

Lactate oxidase nanocapsule and preparation method thereof

[0001] Cross-references

[0002] This invention claims priority from an application filed in China with application number 202311281613.2, filed on October 6, 2023, entitled “A Lactate Oxidase Nanocapsule and Preparation Method Thereof.” The aforementioned application is incorporated herein by reference in its entirety as a part thereof. Technical Field

[0003] The invention relates to a lactate oxidase nanocapsule and a preparation method thereof, and belongs to the technical field of biomedicine. Background Art

[0004] Immunotherapy, which harnesses the host's own immune system to attack tumors, has become an important cancer treatment option alongside surgery, chemotherapy, and radiotherapy. However, immunotherapy does not show a positive response in all cancer patients, primarily due to the hindrance of the patient's tumor's immunosuppressive microenvironment.

[0005] Elevated lactate concentrations in tumors are considered a marker of cancer progression and metastasis. Within tumors, tumor cells rapidly undergo glycolysis and produce excessive amounts of lactate, creating a lactate-rich microenvironment. This high-lactate microenvironment interferes with the proliferation of effector T cells, which primarily rely on glycolysis, and their production of proinflammatory cytokines. However, tumor-resident regulatory T cells can utilize lactate as a carbon source, supporting their suppressive function and contributing to the immunosuppression of the tumor microenvironment. Therefore, reducing lactate concentrations in the tumor microenvironment is a key issue in alleviating tumor immunosuppression and improving effector T cell function. Activating immune cells in the tumor microenvironment is another key issue in cancer immunotherapy. Hydrogen peroxide can effectively activate the NF-κB signaling pathway and increase the secretion of proinflammatory cytokines, activating and directing immune cell migration to sites of hydrogen peroxide enrichment. Hydrogen peroxide production may be key to addressing immune cell activation.

[0006] Summary of the Invention

[0007] The present invention addresses the core issues of tumor immunotherapy and provides a lactate oxidase nanocapsule and a preparation method thereof.

[0008] In a first aspect, the present invention provides a lactate oxidase (LOx) nanocapsule. A monomer and a cross-linking agent are used to form a cross-linked network shell on the surface of the lactate oxidase, and the lactate oxidase is encapsulated in the shell to form a lactate oxidase nanocapsule.

[0009] Preferably, the monomer is an acrylate substance, more preferably, the monomer includes one or more of poly(ethylene glycol) methyl ether acrylate, 2-methacryloyloxyethyl phosphorylcholine (MPC), N-(3-aminopropyl) methacrylamide (APM), acrylamide (AAM), vinyl pyrrolidone, acryloyloxyethyl trimethylammonium chloride, methacryloyloxyethyl trimethylammonium chloride, [2-(methacryloyloxy)ethyl] dimethyl-(3-sulfonic acid propyl) ammonium hydroxide, 3-[[2-(methacryloyloxy)ethyl] dimethylammonium] propionate, and 3-[(3-acrylamidopropyl) dimethylammonium] propionate;

[0010] Preferably, the cross-linking agent is one or more of N,N'-methylenebisacrylamide (BIS), glycerol dimethacrylate and triblock polylactic acid polyethylene glycol dimethacrylate.

[0011] Preferably, the molar ratio of the lactate oxidase, monomer and cross-linking agent is 1: (4000-20000): (400-2000), more preferably, the molar ratio of the lactate oxidase, monomer and cross-linking agent is 1: (10000-20000): (500-2000).

[0012] In a specific embodiment, the monomers include 2-methacryloyloxyethyl phosphorylcholine (MPC) and N-(3-aminopropyl)methacrylamide (APM), and the cross-linking agent includes N,N'-methylenebisacrylamide (BIS).

[0013] Preferably, the MPC:APM is (10000-20000):(500-2000).

[0014] In a specific embodiment, the molar ratio of lactate oxidase:MPC:APM:BIS is 1:(10000-20000):(500-2000):(500-2000). Preferably, the molar ratio of lactate oxidase:MPC:APM:BIS is 1:16000:500:1000.

[0015] The particle size range of the lactate oxidase nanocapsules is 10-200 nm, and can be any range or value within the above range, for example, 10-150 nm, 10-100 nm, 10-80 nm, 20-60 nm, 20-50 nm, 20 nm, 30 nm, 40 nm, etc.

[0016] Preferably, the lactate oxidase nanocapsules have a lower Zeta potential than that of non-nanoencapsulated lactate oxidase, preferably, for example, lower than 5 mV. More preferably, the Zeta potential of the lactate oxidase nanocapsules is 0.5-5 mV, more preferably any value or range within the above range, such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, etc.

[0017] In a second aspect, the present invention provides a method for preparing the above-mentioned lactate oxidase nanocapsules, which comprises using monomers and a cross-linking agent to form a cross-linked network shell on the surface of lactate oxidase, and encapsulating the lactate oxidase in the shell to form lactate oxidase nanocapsules.

[0018] Preferably, the preparation method comprises:

[0019] (1) N-hydroxysuccinimide acrylate (NAS) was dissolved in dimethyl sulfoxide (DMSO) at room temperature;

[0020] (2) Lactate oxidase and DMOS solution of N-hydroxysuccinimide acrylate were mixed with phosphate buffer at room temperature and reacted at 4°C;

[0021] (3) mixing the lactate oxidase, monomer, cross-linking agent and initiator in step (2) with a phosphate buffered saline solvent at room temperature;

[0022] (4) At 4°C, a free radical polymerization reaction is initiated by an initiator on the surface of lactate oxidase, encapsulating the lactate oxidase in a network shell formed by a high molecular weight polymer to form lactate oxidase nanocapsules;

[0023] (5) The obtained reaction product was dialyzed to obtain lactate oxidase nanocapsules n(LOx).

[0024] Furthermore, in step (3) and step (4): lactate oxidase is dissolved in phosphate buffered saline solution, and then monomers, cross-linking agents, and initiators are added in sequence, and then free radical polymerization reaction is carried out at 4°C.

[0025] In the above scheme, the monomer is an acrylic acid ester substance, including one or more of poly (ethylene glycol) methyl ether acrylate, 2-methacryloyloxyethyl phosphorylcholine, N-(3-aminopropyl) methacrylamide, acrylamide, vinyl pyrrolidone, acryloyloxyethyl trimethylammonium chloride, methacryloyloxyethyl trimethylammonium chloride, [2-(methacryloyloxy)ethyl] dimethyl-(3-sulfonic acid propyl) ammonium hydroxide, 3-[[2-(methacryloyloxy)ethyl] dimethylammonium] propionate, and 3-[(3-acrylamidopropyl) dimethylammonium] propionate.

[0026] In the above solution, the cross-linking agent is one or more of N,N'-methylenebisacrylamide, glycerol dimethacrylate and triblock polylactic acid polyethylene glycol dimethacrylate.

[0027] In the above embodiment, the molar ratio of N-hydroxysuccinimide acrylate to lactate oxidase is 1:1-50. More preferably, the molar ratio of N-hydroxysuccinimide acrylate to lactate oxidase is 1:1-20.

[0028] In the above solution, the molar ratio of lactate oxidase, monomer and cross-linking agent is 1:4000-20000:400-2000. More preferably, the molar ratio of lactate oxidase, monomer and cross-linking agent is 1:10000-20000:500-2000.

[0029] In a specific embodiment, the monomers include 2-methacryloyloxyethyl phosphorylcholine (MPC) and NN-(3-aminopropyl) methacrylamide (APM), and the cross-linking agent includes N,N'-methylenebisacrylamide (BIS).

[0030] Preferably, the MPC:APM is (10000-20000):(500-2000).

[0031] In a specific embodiment, the molar ratio of lactate oxidase:MPC:APM:BIS is 1:(10000-20000):(500-2000):(500-2000). Preferably, the molar ratio of lactate oxidase:MPC:APM:BIS is 1:16000:500:1000.

[0032] The particle size range of the lactate oxidase nanocapsules is 10-200 nm, and can be any range or value within the above range, for example, 10-150 nm, 10-100 nm, 10-80 nm, 20-60 nm, 20-50 nm, 20 nm, 30 nm, 40 nm, etc.

[0033] Preferably, the lactate oxidase nanocapsules have a lower Zeta potential than that of non-nanoencapsulated lactate oxidase, preferably, for example, lower than 5 mV. More preferably, the Zeta potential of the lactate oxidase nanocapsules is 0.5-5 mV, more preferably any value or range within the above range, such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, etc.

[0034] In a third aspect, the present invention provides an application of the lactate oxidase nanocapsules, comprising:

[0035] 1) Use of the lactate oxidase nanocapsules for preparing a drug for reducing the concentration of lactic acid in tumors and releasing hydrogen peroxide in situ,

[0036] 2) Use of the lactate oxidase nanocapsules in preparing drugs for inhibiting or treating tumors,

[0037] 3) Use of the lactate oxidase nanocapsules for preparing drugs for activating immune cells,

[0038] 4) Use of the lactate oxidase nanocapsules for preparing immunotherapy drugs, or

[0039] 5) Use of the lactate oxidase nanocapsules for preparing a drug for combined use with a tumor immunotherapy inhibitor, wherein the drug is used to treat tumors.

[0040] Preferably, the tumor immunotherapy inhibitor includes:

[0041] Cytostatics, such as vemurafenib (BRAF inhibitor) and cobimetinib (MEK inhibitor), induce immunogenic cell death (ICD) that can trigger the activation of antitumor T cell responses;

[0042] VEGF–VEGFR inhibitors, such as axitinib (VEGFR tyrosine kinase inhibitor) and bevacizumab (VEGF antibody), promote antigen presentation and recruitment of immunosuppressive cells by blocking angiogenesis;

[0043] Cytokine and chemokine pathway blockers, such as pecidinib (CSF1R-TK inhibitor), which block cytokine pathways and / or suppress T cell immunity in the process of cancer-promoting inflammation; and / or,

[0044] Immune checkpoint inhibitors (ICIs), such as PD-L1 antibodies, CA-170 and GS-4224 (PDL1–PD1 small molecule inhibitors), Pembrolizumab and Nivolumab (antibodies, PD-1 inhibitors), Atezolizumab and Durvalumab (antibodies, PD-L1 inhibitors), and Ipilimumab (antibody, CTLA-4 inhibitor). Preferably, the tumor in the above 2) is any malignant tumor, including solid tumors, hematologic tumors, etc., such as breast cancer, skin cancer, lymphoma, etc.

[0045] The immune cells in the above 3) include T cells, and the drug can increase the expression of CD107a, CD69 and / or IFN-γ in T cells.

[0046] Preferably, the lactate oxidase nanocapsules can be prepared into a drug form for subcutaneous administration, intravenous administration, and intratumoral injection.

[0047] In a fourth aspect, the present invention provides a drug comprising any one of the above-mentioned lactate oxidase nanocapsules.

[0048] Preferably, the drug can be used for:

[0049] 1) Reduce the concentration of lactate in tumors and release hydrogen peroxide in situ,

[0050] 2) inhibit or treat tumors,

[0051] 3) Activate immune cells, or,

[0052] 4) Immunotherapy.

[0053] Preferably, the tumor in the above 2) is any malignant tumor, including solid tumors, hematological tumors, etc., such as breast cancer, skin cancer, lymphoma, etc.

[0054] The immune cells in the above 3) include T cells, and the drug can increase the expression of CD107a, CD69 and / or IFN-γ in T cells.

[0055] Preferably, the drug further comprises the tumor immunotherapy inhibitor, and more preferably, the tumor immunotherapy inhibitor comprises:

[0056] Cytostatics, such as vemurafenib (BRAF inhibitor) and cobimetinib (MEK inhibitor), induce immunogenic cell death (ICD) that can trigger the activation of antitumor T cell responses;

[0057] VEGF–VEGFR inhibitors, such as axitinib (VEGFR tyrosine kinase inhibitor) and bevacizumab (VEGF antibody), promote antigen presentation and recruitment of immunosuppressive cells by blocking angiogenesis;

[0058] Cytokine and chemokine pathway blockers, such as pecidinib (CSF1R-TK inhibitor), which block cytokine pathways and / or suppress T cell immunity in the process of cancer-promoting inflammation; and / or,

[0059] Immune checkpoint inhibitors (ICIs), such as PD-L1 antibodies, CA-170 and GS-4224 (PDL1–PD1 small molecule inhibitors), Pembrolizumab and Nivolumab (antibodies, PD-1 inhibitors), Atezolizumab and Durvalumab (antibodies, PD-L1 inhibitors), Ipilimumab (antibody, CTLA-4 inhibitor), etc.

[0060] The drug can be prepared into a drug form for subcutaneous administration, intravenous administration and intratumoral injection.

[0061] Technical effects of the present invention:

[0062] The present invention adopts aqueous in situ free radical polymerization, and utilizes electrostatic and hydrophobic forces to adsorb monomers and cross-linking agents around lactate oxidase molecules, forming a cross-linked network to encapsulate lactate oxidase, thereby preparing a lactate oxidase nanocapsule. The lactate oxidase nanocapsule can reduce the concentration of lactic acid in the tumor and release hydrogen peroxide in situ, thereby alleviating tumor immunosuppression, recruiting immune cells to the tumor microenvironment, and improving the immune therapeutic effect. The capsule of the present invention enhances the stability of lactate oxidase, ensuring that it can efficiently reach the target site and exert its drug effect after persistent circulation in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG1 is an SDS-PAGE characterization diagram of lactate oxidase nanocapsules.

[0064] FIG2 is a HPLC characterization diagram of lactate oxidase nanocapsules.

[0065] FIG3 shows the particle size distribution and Zeta potential of lactate oxidase nanocapsules: (A) particle size distribution of lactate oxidase (Lox) and lactate oxidase nanocapsules n(LOx); (B) Zeta potential of LOx and n(LOx).

[0066] Figure 4 shows that lactate oxidase nanocapsules reduce the intracellular lactate concentration and increase the hydrogen peroxide concentration in the culture medium: (A) Lactate oxidase nanocapsules (n(LOx)) can reduce the lactate concentration produced by cells; (B) n(LOx) can increase the hydrogen peroxide concentration in the cell culture medium.

[0067] Figure 5 shows that lactate oxidase nanocapsules regulate T cell activation: (A) CD107a expression in CD4 T cells; (B) CD107a expression in CD8 T cells; (C) CD69 expression in CD4 T cells; (B) CD69 expression in CD8 T cells.

[0068] FIG6 shows that lactate oxidase nanocapsules regulate cytokine expression: (A) IFN-γ expression in CD4 T cells; (B) IFN-γ expression in CD8 T cells.

[0069] Figure 7 shows the activation of chimeric antigen receptor (CAR) T cells by lactate oxidase nanocapsules: (A) the survival rate of cancer cells after the addition of CAR-T cells and CAR-T cells and n(LOx); (B) the expression level of CD69 in CAR-T cells after the addition of n(LOx).

[0070] FIG8 shows the inhibition of breast cancer growth in mice by lactate oxidase nanocapsules: (A) tumor growth curve; (B) tumor mass.

[0071] Figure 9 shows the biosafety of lactate oxidase nanocapsules: changes in the levels of liver markers ALT / AST (A) and kidney markers CREA / BUN (B) in the serum of mice in each group. Note: The dotted lines in the figure represent the normal range.

[0072] Figure 10 shows the combined effect of lactate oxidase nanocapsules (n(LOx)) and anti-PD-L1 (aPD-L1) in mouse skin cancer B16F10: (A) tumor growth curve, (B) survival curve. DETAILED DESCRIPTION

[0073] The following are examples of the present invention. It is necessary to point out that the examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Those skilled in the art may make some non-essential improvements and adjustments based on the above contents of the present invention.

[0074] 1 Preparation of lactate oxidase nanocapsules

[0075] 1.1 Preparation of nanocapsules

[0076] (1) Reaction of Lactate Oxidase (LOx) and N-Hydroxysuccinimidyl acrylate (NAS): In a 50 mL test tube, lactate oxidase was first dissolved in 50 mM phosphate buffer (PB) at pH 8.0. NAS dissolved in dimethyl sulfoxide (DMSO) was then added at a ratio of LOx:NAS = 1:20 (n / n). The reaction was allowed to proceed at 4°C for two hours.

[0077] (2) Removal of unreacted NAS: Take a 30 kDa dialysis bag of appropriate length, place the reacted LOx-NAS into the dialysis bag, and clamp the ends of the dialysis bag with clamps. Place the dialysis bag in a beaker containing 1× PBS solution and place it in a 4°C refrigerator for dialysis. Change the solution every 6-12 hours to remove unreacted NAS. Dialysis should be continued for 24 hours.

[0078] (3) Synthesis of n(LOx): According to the LOx concentration, the following formula was used: LOx: 2-methacryloyloxyethyl phosphorylcholine (MPC) (monomers can also be replaced by poly(ethylene glycol) methyl ether acrylate (PEG-Acrylate), vinyl pyrrolidone (NVP)): N-(3-aminopropyl) methacrylamide (APM): BIS = 1:16000:500:1000 (n / n) (MPC (PEG-Acrylate, NVP) ratio can be adjusted to 10000-20000, AP Add 1× PBS, LOx, MPC, APM, and BIS to a test tube in this order (the ratio of LOx to MPC is adjustable from 500-2000, and the ratio of BIS is adjustable from 500-2000). Vortex to mix thoroughly. Then, add ammonium persulfate (APS) at a ratio of LOx to APS of 1:500 (n / n). Vortex again to mix thoroughly. Finally, add tetramethylethylenediamine (TEMED) at a ratio of LOx to TEMED of 1:1000 (n / n). Free radical polymerization reaction was carried out at 4°C for 2 hours.

[0079] (4) Purification of n(LOx): Unreacted n(LOx) was removed using an ultracentrifugal filter with a molecular weight cutoff of 50 kDa. The wash buffer was 10 mM phosphate buffer solution (PBS, pH 7.2-7.4). The resulting n(LOx) was then stored at 4°C for subsequent experimental use.

[0080] 1.2 Purification of nanocapsules

[0081] The crude nanocapsules n(LOx) were filtered at 4° C. using an ultracentrifugal filter with a molecular weight cutoff of 50 KDa to remove unreacted monomers and cross-linking agents. The washing buffer was 10 mM phosphate buffer (pH 7.2-7.4).

[0082] 2 Functional evaluation of lactate oxidase nanocapsules

[0083] 2.1 SDS-PEGE analysis of nanocapsules

[0084] A 0.5 mg / ml LOx solution and n(LOx) nanocapsule solution were mixed with 5× loading buffer and incubated at 70°C for 15 minutes. The mixture was loaded onto the wells of a 10% (w / v) sodium dodecyl sulfate polyacrylamide gel and electrophoresed at 120V for 60 minutes. The gel was stained with Coomassie Brilliant Blue R-250 at room temperature for 1 hour and then destained overnight in 10% glacial acetic acid / 30% methanol. After destaining, the gel was imaged.

[0085] 2.2 HPLC Characterization of Nanocapsules

[0086] The characterization of Lox and n(LOx) was performed using an Agilent high performance liquid chromatograph with BioCore SEC-300 after loading the sample with 150 mM PB buffer as the mobile phase.

[0087] 2.3 Particle size distribution and zeta potential analysis of nanocapsules

[0088] The sample was diluted to 0.1 mg / mL and filtered through a 0.22 μm filter membrane. Zeta potential was measured using a Malvern Zetasizer Nano ZSE instrument.

[0089] 2.4 Effects of Nanocapsules on Cancer Cells

[0090] 5x 10 3 4T1 cells were seeded at a density of 100 μL / well in a 96-well plate and 5 μg / mL of n(LOx) was added. Cell culture medium was collected at different times. Lactate concentration was determined using the lactate dehydrogenase (LDH) assay. H2O2 production in the cell culture medium was detected using H2DCFDA.

[0091] 2.5 Nanocapsules activate T cells

[0092] 1x 10 6 10 mM lactic acid and 0.05 μg / mL n(LOx) were added to PBMCs cells at a concentration of 1 μg / mL, and immunostaining was performed after incubation for 24 hours. Flow cytometry was used to detect the expression of T cell surface proteins and the expression of cytokines in immune cells.

[0093] 2.6 Nanocapsules activate chimeric antigen receptor (CAR) T cells

[0094] 1x 10 6 CD19CAR-T cells / mL and 1*10 6 / mL 2F7B cell lymphoma cells were co-incubated, and then 0.05μg / mL n(LOx) was added. After incubation for 24 hours, immunostaining was performed, and the expression of T cell surface proteins and the survival of B cell lymphoma were detected by flow cytometry.

[0095] Effects of 2.7 nanocapsules on breast cancer growth in mice

[0096] 5x10 54T1 cells were mixed with RPMI / Matrigel and injected subcutaneously into the second mammary gland of female BALB / c mice. Seven days after tumor injection, the mice were randomly divided into three groups and treated intratumorally with PBS, LOx (0.5 mg / kg), or n(LOx) (0.5 mg / kg) every three days. Tumor size and tumor size were analyzed.

[0097] 2.8 Biosafety of Nanocapsules

[0098] The mice were randomly divided into groups and treated with different doses of n(LOx) intratumorally every 3 days. The levels of ALT, AST, CREA and BUN in the serum of the mice were detected.

[0099] 2.9 Nanocapsules combined with immune checkpoint blockers for tumor treatment

[0100] Forty mice were randomly divided into four groups of 10 mice each. 400,000 mouse B16F10 skin cancer cells were injected subcutaneously into each C57BL / 6J mouse on day 0. In the first group, n(LOx) (0.5 mg / kg) was injected intratumorally on days 7 and 9 after injection. In the second group, an equal volume of phosphate-buffered saline (PBS) was injected intratumorally on days 7 and 9 after injection. In the third group, aPD-L1 (100 μg / mouse) was injected intraperitoneally on days 9, 12, 16, and 19 after injection. For the fourth group of mice, n(LOx) was injected intratumorally (0.5 mg / kg) on ​​the 7th and 9th days after the injection of mouse skin cancer cells, and aPD-L1 (100 μg / mouse) was injected intraperitoneally on the 9th, 12th, 16th and 19th days after the injection of mouse skin cancer cells.

[0101] 3 Experimental results

[0102] 3.1 SDS-PEGE analysis results of nanocapsules

[0103] Lactate oxidase (LOx) was encapsulated in a polymer shell composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), N-(3-aminopropyl) methacrylamide (APM) and N,N'-methylenebisacrylamide (BIS) to obtain lactate oxidase nanocapsules n(LOx).

[0104] As shown in Figure 1 , compared with lactate oxidase that was not nanoencapsulated, lactate oxidase nanocapsules n(LOx) were retained in the sample wells, indicating that their volume became larger after nanoencapsulation and they could not migrate quickly in the SDS-PEGE gel.

[0105] As shown in Figure 2 , the retention time of lactate oxidase nanocapsules n(LOx) in the chromatographic column is shorter than that of LOx (the left curve is n(LOx)), indicating that the molecular weight of LOx increases after nanoencapsulation, further proving the successful encapsulation of LOx.

[0106] 3.2 HPLC characterization results of nanocapsules

[0107] No UV absorption signal of LOx appeared in the lactate oxidase nanocapsule n(LOx) sample, proving that the lactate oxidase nanocapsule n(LOx) was completely separated from lactate oxidase (LOx), which was consistent with the SDS-PAGE results in Figure 1.

[0108] 3.3 Particle size distribution and Zeta potential analysis results of nanocapsules

[0109] Figure 3 shows that the particle size of lactate oxidase nanocapsules n(LOx) is about 20 nm (Figure 3A, where n(LOx) is the right curve) and the Zeta potential is 1 mV (Figure 3B), which is significantly different from that of lactate oxidase (LOx) that has not been nanoencapsulated (7 nm and -5.4 mV).

[0110] The above results indicate that the monomer and cross-linker form a cross-linked network on the surface of lactate oxidase and successfully encapsulate the lactate oxidase.

[0111] 3.4 Effect of Lactate Oxidase Nanocapsules on Eliminating Lactic Acid and Producing Hydrogen Peroxide in Tumor Cells

[0112] To verify the effectiveness of lactate oxidase in eliminating lactate and producing hydrogen peroxide, lactate oxidase nanocapsules (LOx) were added to mouse breast cancer cells and co-incubated. The lactate concentration in the cell culture medium decreased rapidly, and hydrogen peroxide was produced in the culture medium (Figure 4).

[0113] Figure 4A shows that both lactate oxidase (LOx, 5 μg / mL) and its nanocapsules (n(LOx), 5 μg / mL) were able to reduce lactate concentrations in cell culture medium. Within one hour, lactate concentrations in the cell culture medium decreased from approximately 14 mM to approximately 5 mM, with n(LOx) being slightly more effective than LOx (Figure 4A, right bar graph shows n(LOx)). Furthermore, the hydrogen peroxide concentration in the culture medium showed an upward trend. Twenty-four hours after addition of LOx or n(LOx), the fluorescence concentration of the hydrogen peroxide probe increased by 7-fold and 10-fold, respectively (Figure 4B). Compared to LOx, nanocapsules exhibited greater stability after encapsulation, produced more hydrogen peroxide in the culture medium, and more effectively eliminated lactate.

[0114] 3.5 Effect of lactate oxidase nanocapsules on T cell activation

[0115] To verify the effect of lactate oxidase on immune cells, lactate oxidase nanocapsules (LOx) were co-incubated with PBMCs in a 10mM lactate environment. After the addition of lactate oxidase nanocapsules (LOx), T cell activation markers CD107a and CD69 were significantly increased (Figure 5, Tables 1 and 2):

[0116] Table 1: Detection results of CD107a, an activation marker of T cells, under the action of lactate oxidase nanocapsules n(LOx)

[0117] Table 2: Detection results of CD69, an activation marker of T cells, under the action of lactate oxidase nanocapsules n(LOx)

[0118] Interferon-γ (IFN-γ) was also secreted in large quantities ( Figure 6 and Table 3 ), demonstrating that the addition of lactate oxidase nanocapsules n(LOx) could activate immune cells.

[0119] Table 3: Detection results of interferon-γ secreted by T cells under the action of lactate oxidase nanocapsules n(LOx)

[0120] 3.6 Effect of lactate oxidase nanocapsules on the activation of chimeric antigen receptor (CAR) T cells

[0121] In addition, the co-incubation experiment of chimeric antigen receptor (CAR) T cells and lymphoma cells also showed that the addition of lactate oxidase nanocapsules n (LOx) can activate T cells and promote their ability to kill tumors (Figure 7 and Table 4).

[0122] Table 4: Effects of lactate oxidase nanocapsules (LOx) on the activation of chimeric antigen receptor (CAR) T cells

[0123] These results demonstrate that Lactate Oxidase Nanocapsules (LOx) can eliminate lactate, produce hydrogen peroxide, and activate immune cells, particularly T cells. Due to their immune system regulatory properties, Lactate Oxidase Nanocapsules (LOx) have broad implications for the treatment of immune-related diseases associated with lactate accumulation, such as arthritis, diabetic nephropathy, cachexia, and tumors.

[0124] 3.7 Inhibitory effect of lactate oxidase nanocapsules on breast cancer in mice

[0125] The tumor-suppressing effect of lactate oxidase nanocapsules n(LOx) was further investigated in a mouse model of breast cancer. As shown in Figure 8, both lactate oxidase (LOx) and lactate oxidase nanocapsules n(LOx) significantly inhibited tumor growth compared to the group injected with PBS alone (Figure 8A, top to bottom, curves represent PBS, Lox, and n(LOx)). Due to the protective effect of nanoencapsulation on lactate oxidase (LOx), lactate oxidase nanocapsules n(LOx) were able to significantly and effectively inhibit tumor growth compared to unencapsulated lactate oxidase (LOx).

[0126] 3.8 Biosafety of Lactate Oxidase Nanocapsules

[0127] In addition, the liver (AST) and kidney (CREA) function indicators of mice were tested, and the results showed that lactate oxidase nanocapsules n(LOx) did not affect the liver and kidney functions of mice (Figure 9, the bar graph from left to right is Blank, 0.1 mg / kg, 0.3 mg / kg, and 0.5 mg / kg), indicating that lactate oxidase nanocapsules n(LOx) have good biosafety and can be used as an immune preparation in tumor immunotherapy.

[0128] 3.9 Lactate oxidase nanocapsules combined with immune checkpoint blockers have therapeutic effects on tumors

[0129] In a skin cancer mouse model, the tumor inhibitory effect of lactate oxidase nanocapsules n(LOx) combined with immune checkpoint blockers was further studied.

[0130] As shown in Figure 10A, aPD-L1 alone was unable to inhibit B16F10 tumor growth, exhibiting essentially the same exponential growth trend as the control group (Figure 10A, top to bottom, curves represent PBS, aPD-L1, n(LOx), and n(LOx) / aPD-L1). n(LOx) effectively inhibited tumor growth, but tumors resumed growth about a week after cessation of injection. In contrast, the combination of n(LOx) and aPD-L1 significantly inhibited tumor growth. The survival curves in Figure 10B also demonstrate that the survival rate of the aPD-L1 group alone dropped to approximately 80% on the 15th day and to 0% on the 18th day, the survival rate of the n(LOx) group alone dropped to approximately 70% on the 21st day, and the survival rate of the n(LOx) / aPD-L1 group remained at 100%. This shows that n(LOx) significantly enhances the therapeutic effect of aPD-L1 in mouse skin cancer (the curves in Figure 10B are PBS, aPD-L1, n(LOx), and n(LOx) / aPD-L1 from left to right).

Claims

1. A lactate oxidase nanocapsule, characterized in that: A cross-linked network shell is formed on the surface of lactate oxidase using monomers and a cross-linking agent, and the lactate oxidase is wrapped in the shell to form a lactate oxidase nanocapsule.

2. The lactate oxidase nanocapsule according to claim 1, characterized in that: The monomer is an acrylate substance. Preferably, the monomer includes poly(ethylene glycol) methyl ether acrylate, 2-methacryloyloxyethyl phosphorylcholine (2-Methacryloyloxyethyl phosphorylcholine (MPC), N-(3-aminopropyl)methacrylamide (APM), acrylamide (AAM), vinyl pyrrolidone, acryloyloxyethyl trimethylammonium chloride, methacryloyloxyethyl trimethylammonium chloride, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium] propionate, 3-[(3-acrylamidopropyl)dimethylammonium] propionate. Preferably, the monomer includes 2-methacryloyloxyethyl phosphorylcholine (MPC) and N-(3-aminopropyl)methacrylamide (APM). More preferably, the MPC:APM is (10000-20000):(500-2000); The crosslinking agent is one or more of N,N'-methylenebisacrylamide (BIS), glycerol dimethacrylate and triblock polylactic acid polyethylene glycol dimethacrylate.

3. The lactate oxidase nanocapsule according to any one of claims 1-2, characterized in that: The particle size of the lactate oxidase nanocapsule is in the range of 10-200 nm.

4. The lactate oxidase nanocapsule according to any one of claims 1 to 3, characterized in that: The molar ratio of the lactate oxidase, the monomer and the cross-linking agent is 1: (4000-20000): (400-2000). Preferably, the molar ratio of the lactate oxidase, the monomer and the cross-linking agent is 1: (10000-20000): (500-2000).

5. The method for preparing lactate oxidase nanocapsules according to any one of claims 1 to 4, characterized in that: The preparation method comprises the steps of forming a cross-linked network shell on the surface of lactate oxidase using monomers and a cross-linking agent, and encapsulating the lactate oxidase in the shell to form a lactate oxidase nanocapsule.

6. The preparation method according to claim 5, characterized in that The preparation method comprises: (1) dissolving N-hydroxysuccinimide acrylate (NAS) in dimethyl sulfoxide (DMSO) at room temperature; (2) mixing lactate oxidase and DMOS solution of N-hydroxysuccinimide acrylate with phosphate buffer at room temperature and reacting at 4°C; (3) mixing the lactate oxidase, monomer, cross-linking agent and initiator of step (2) with a phosphate buffered saline solvent at room temperature; (4) At 4°C, a free radical polymerization reaction was initiated by an initiator on the surface of lactate oxidase to encapsulate the lactate oxidase. Wrapped in a network shell formed by a high molecular polymer, lactate oxidase nanocapsules are formed; (5) The obtained reaction product is dialyzed to obtain purified lactate oxidase nanocapsules n(LOx).

7. The preparation method according to any one of claims 5-6, characterized in that: The molar ratio of the N-hydroxysuccinimide acrylate to the lactate oxidase is 1:1-50, preferably, the molar ratio of the N-hydroxysuccinimide acrylate to the lactate oxidase is 1:1-208.

8. The use of the lactate oxidase nanocapsules according to any one of claims 1 to 4, characterized in that: The applications include: 1) Use of the lactate oxidase nanocapsules for preparing a drug for reducing the lactic acid concentration in tumors and releasing hydrogen peroxide in situ, 2) Use of the lactate oxidase nanocapsules for preparing drugs for inhibiting or treating tumors, 3) Use of the lactate oxidase nanocapsules for preparing drugs for activating immune cells, 4) Use of the lactate oxidase nanocapsules for preparing immunotherapy drugs, or, 5) Use of the lactate oxidase nanocapsules for preparing a drug for combined use with a tumor immunotherapy inhibitor, wherein the drug is used to treat tumors.

9. The use according to claim 8, characterized in that: The tumor immunotherapy inhibitor is selected from: one or more of: cell growth inhibitors, VEGF-VEGFR inhibitors, cytokine and chemokine pathway blockers, and immune checkpoint inhibitors (ICIs).

10. The use according to claim 8 or 9, characterized in that: The lactate oxidase nanocapsules are prepared into a drug form for subcutaneous administration, intravenous administration and intratumoral injection.

11. A drug, characterized in that The drug includes the lactate oxidase nanocapsules described in any one of claims 1-4. Preferably, the drug also includes a tumor immunotherapy inhibitor. More preferably, the tumor immunotherapy inhibitor is selected from: one or more of: cell growth inhibitors, VEGF-VEGFR inhibitors, cytokine, chemokine pathway blockers, and immune checkpoint inhibitors (ICIs).

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