Lactate oxidase nanocapsule and preparation method thereof

Abstract

A lactate oxidase nanocapsule and a preparation method thereof belong to the field of biomedical technology. The present invention uses poly (ethylene glycol) methyl ether acrylate, 2-methacryloyloxyethyl phosphorylcholine, N-(3-aminopropyl) methacrylamide and acrylamide as monomers, N, N'-methylenebisacrylamide, dimethacrylate glyceride and triblock polylactic acid polyethylene glycol dimethacrylate as cross-linking agents, and utilizes free radical polymerization to synthesize a nanometer-thick polymer shell in situ on the surface of lactate oxidase to construct lactate oxidase nanocapsules. Lactate oxidase nanocapsules can eliminate lactic acid and release immunostimulants, eliminate immunosuppression, and can be used in combination with immune checkpoint blockers to improve the immune efficacy of immune checkpoint blockers.

Description

Technical Field

[0001] The invention relates to a lactate oxidase nanocapsule and a preparation method thereof, belonging to the technical field of biomedicine. Background Art

[0002] Immunotherapy, which uses the host's own immune system to attack tumors, has become another important cancer treatment method in addition to surgery, chemotherapy, and radiotherapy. However, immunotherapy does not show a positive response in all tumor patients, mainly due to the hindrance of the patient's tumor immunosuppressive microenvironment.

[0003] Increased lactate concentration in tumors is considered an indicator of cancer progression and metastasis. In tumors, tumor cells can rapidly undergo glycolysis and produce excessive lactate, thus forming a lactate-rich microenvironment. This high-lactate microenvironment interferes with the proliferation of effector T cells that mainly rely on glycolysis and their production of proinflammatory cytokines. However, tumor-resident regulatory T cells can use lactate as a carbon source, supporting their suppressive function and promoting immunosuppression in the tumor microenvironment. Therefore, how to reduce the lactate concentration in the tumor microenvironment is a core issue in alleviating tumor immunosuppression and improving effector T cell function. How to activate immune cells in the tumor microenvironment is another core issue in tumor immunotherapy. Hydrogen peroxide can effectively activate the NF-κB signaling pathway, increase the secretion of proinflammatory cytokines, activate and guide immune cells to migrate to hydrogen peroxide-rich sites. The production of hydrogen peroxide may be the key to solving the problem of immune cell activation. Summary of the invention

[0004] Aiming at the core problem of tumor immunotherapy, the present invention provides a lactate oxidase nanocapsule and a preparation method thereof.

[0005] A lactate oxidase nanocapsule, characterized in that a monomer and a cross-linking agent form a cross-linked network shell on the surface of lactate oxidase, and the lactate oxidase is encapsulated in the shell. The particle size of the lactate oxidase nanocapsule ranges from 10 to 100 nm, and most of them are 20 nm.

[0006] The present invention provides a lactate oxidase nanocapsule and a preparation method thereof, wherein the preparation method is as follows:

[0007] (1) dissolving N-hydroxysuccinimide acrylate (NAS) in dimethyl sulfoxide (DMSO) at room temperature;

[0008] (2) mixing lactate oxidase and DMOS solution of N-hydroxysuccinimide acrylate with phosphate buffer at room temperature and reacting at 4°C;

[0009] (3) mixing the lactate oxidase, monomer, cross-linking agent and initiator in step (2) with a phosphate buffered saline solvent at room temperature;

[0010] (4) At 4°C, a free radical polymerization reaction is initiated by an initiator on the surface of lactate oxidase, and the lactate oxidase is encapsulated in a network shell formed by a high molecular weight polymer to form lactate oxidase nanocapsules;

[0011] (5) The obtained reaction product is dialyzed to obtain lactate oxidase nanocapsules n(LOx).

[0012] Further steps (3) and (4): dissolving lactate oxidase in phosphate buffered saline solution, then sequentially adding monomers, crosslinking agents, and initiators, and then conducting free radical polymerization at 4°C.

[0013] 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, acryloyloxyethyltrimethylammonium chloride, methacryloyloxyethyltrimethylammonium chloride, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl)ammonium hydroxide, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, and 3-[(3-acrylamidopropyl)dimethylammonium]propionate.

[0014] In the above scheme, the crosslinking agent is one or more of N,N'-methylenebisacrylamide, glycerol dimethacrylate and triblock polylactic acid polyethylene glycol dimethacrylate. In the above scheme, the molar ratio of N-hydroxysuccinimide acrylate to lactate oxidase is 1:1-50. It is further preferred that the molar ratio of N-hydroxysuccinimide acrylate to lactate oxidase is 1:1-20.

[0015] In the above scheme, 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.

[0016] The nanocapsules can eliminate lactic acid and release hydrogen peroxide. The lactate oxidase nanocapsules can be used to prepare drugs that reduce the lactic acid concentration in tumors and release hydrogen peroxide in situ, can be used to prepare drugs that inhibit or treat tumors, and can be used to prepare drugs for activating immune cells.

[0017] The lactate oxidase nanocapsules obtained by the present invention are used for preparing immunotherapy drugs. They are used for preparing drugs used in combination with tumor immunotherapy inhibitors for treating tumors. Tumor immunotherapy inhibitors include: cell growth inhibitors, such as vemurafenib (BRAF inhibitor) and cobimetinib (MEK inhibitor). The immunogenic cell death (ICD) induced by these drugs can trigger the activation of anti-tumor T cell responses; VEGF-VEGFR inhibitors, such as axitinib (VEGFR tyrosine kinase inhibitor) and bevacizumab (VEGF antibody), which promote antigen presentation and immunosuppressive cell recruitment by blocking angiogenesis; cytokines, chemokines Pathway blockers, such as Pecidinib (CSF1R-TK inhibitor), block cytokine pathways and / or inhibit T cell immunity in the process of cancer-promoting inflammation; immune checkpoint inhibitors (ICIs), such as 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).

[0018] The nanocapsules can be prepared into drug forms for subcutaneous administration, intravenous administration, and intratumoral injection.

[0019] The present invention adopts aqueous in-situ free radical polymerization, utilizes electrostatic and hydrophobic forces, makes monomers and cross-linking agents adsorb around lactate oxidase molecules, forms a cross-linked network to wrap lactate oxidase, and prepares a lactate oxidase nanocapsule. The lactate oxidase nanocapsule can reduce the lactic acid concentration in the tumor and release hydrogen peroxide in situ, alleviate tumor immunosuppression, recruit immune cells to the tumor microenvironment, and improve the immunotherapy effect. The capsule of the present invention enhances the stability of lactate oxidase, ensures that it can efficiently reach the target site and exert the drug effect after persistent circulation in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the SDS-PAGE characterization of lactate oxidase nanocapsules.

[0021] Figure 2 HPLC characterization diagram of lactate oxidase nanocapsules.

[0022] Figure 3 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).

[0023] Figure 4Lactate oxidase nanocapsules reduce intracellular lactate concentration and increase hydrogen peroxide concentration in 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 cell culture medium. H2DCFDA is a probe for hydrogen peroxide, which turns into a fluorescent DCF molecule when it encounters hydrogen peroxide. Figure 5 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.

[0024] Figure 6 Lactate oxidase nanocapsules regulate cytokine expression. (A) IFN-γ expression in CD4 T cells. (B) IFN-γ expression in CD8 T cells.

[0025] Figure 7 Lactate oxidase nanocapsules activate chimeric antigen receptor (CAR) T cells. (A) Survival rate of cancer cells after adding CAR-T cells and CAR-T cells and n(LOx). (B) Expression of CD69 in CAR-T cells after adding n(LOx).

[0026] Figure 8 The inhibition of breast cancer growth in mice by lactate oxidase nanocapsules. (A) Tumor growth curve. (B) Tumor mass.

[0027] Fig. 9 The biosafety of lactate oxidase nanocapsules. Changes in the levels of liver index ALT / AST (A) and kidney index CREA / BUN (B) in the serum of mice in each group. Note: The dotted line in the figure represents the normal range.

[0028] Fig.10 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

[0029] 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 cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the above contents of the present invention.

[0030] 1. Preparation of lactate oxidase nanocapsules

[0031] 1.1 Preparation of nanocapsules

[0032] (1) Reaction of lactate oxidase (LOx) and N-hydroxysuccinimidyl acrylate (NAS): Take a 50 mL test tube, first dissolve lactate oxidase in 50 mM phosphate buffer (PB) at pH = 8.0, and then add NAS dissolved in dimethyl sulfoxide (DMSO) at a ratio of LOx:NAS = 1:20 (n / n). After addition, react at 4°C for two hours.

[0033] (2) Removal of unreacted NAS: Take a 30KDa dialysis bag of appropriate length, place the reacted LOx-NAS into the dialysis bag, and clamp the two ends of the dialysis bag with clips. 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 is performed for 24 hours.

[0034] (3) Synthesis of n(LOx): According to the concentration of LOx, 1×PBS, LOx, MPC, and BIS were added to the test tube in the ratio of LOx:2-methacryloyloxyethylphosphocholine (MPC):N-(3-dimethylpropyl)methacrylamide (APM):BIS=1:16000:500:1000 (n / n) (MPC ratio range can be adjusted to 10000-20000, Apm ratio range can be adjusted to 500-2000, and BIS ratio range can be adjusted to 500-2000). After the addition, vortex the system to mix well. Then, APS was added to the test tube in the ratio of LOx:APS=1:500 (n / n). After the addition, vortex the system again. Finally, TEMED was added to the test tube in the ratio of LOx:TEMED=1:1000 (n / n). The reaction was carried out at 4°C for 2h by free radical polymerization.

[0035] (4) Purification of n(LOx): Unreacted n(LOx) was removed using an ultracentrifugal filter with a molecular weight cutoff of 50 kDa, and the washing buffer was 10 mM phosphate buffer solution (PBS, pH 7.2-7.4). The obtained n(LOx) was then stored at 4°C for use in subsequent experiments.

[0036] 1.2 Purification of nanocapsules

[0037] The obtained crude nanocapsule n(LOx) was 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).

[0038] 2 Functional evaluation of lactate oxidase nanocapsules

[0039] 2.1 SDS-PEGE analysis of nanocapsules

[0040] 0.5 mg / ml LOx solution and n(LOx) nanocapsule solution were mixed with 5× loading buffer and incubated at 70°C for 15 min. The mixture was loaded into the sample wells of 10% (w / v) sodium dodecyl sulfate polyacrylamide gel and electrophoresed at 120V for 60 min. The gel was stained with Coomassie Brilliant Blue R-250 staining solution at room temperature for 1 h, and then destained overnight in 10% glacial acetic acid / 30% methanol solution. Gel imaging was performed after destaining.

[0041] 2.2 HPLC characterization of nanocapsules

[0042] The characterization of Lox and n(LOx) was performed using an Agilent HPLC with BioCore SEC-300 after loading with 150 mM PB buffer as the mobile phase.

[0043] 2.3 Particle size distribution and Zeta potential analysis of nanocapsules

[0044] The sample was diluted to 0.1 mg / mL and filtered using a 0.22 μm filter membrane. The zeta potential was measured using a Malvern ZetasizerNano ZSE instrument.

[0045] 2.4 Effects of nanocapsules on cancer cells

[0046] 5x 10 3 4T1 cells at a density of 100uL / well were seeded in a 96-well plate and 5μg / mL of n(LOx) was added. Cell culture medium was collected at different times. Lactate concentration was determined by lactate dehydrogenase (LDH) method. H2O2 generation in cell culture medium was detected by H2DCFDA.

[0047] 2.5 Activation of T cells by nanocapsules

[0048] 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.

[0049] 2.6 Nanocapsules activate chimeric antigen receptor (CAR) T cells 1x 10 6 CD19 CAR-T cells / mL and 1*10 6 / mL 2F7B cell lymphoma cells were co-incubated, and then 0.05μg / mL n(LOx) was added. Immunostaining was performed after 24 hours of co-incubation, and flow cytometry was used to detect the expression of T cell surface proteins and the survival of B cell lymphoma.

[0050] 2.7 Effect of nanocapsules on the growth of breast cancer in mice

[0051] 5x10 5 4T1 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 with PBS, LOx (0.5 mg / kg) or n(LOx) (0.5 mg / kg) intratumorally every 3 days. The tumor size and tumor size of the mice were analyzed.

[0052] 2.8 Biosafety of Nanocapsules

[0053] 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.

[0054] 2.9 Effect of nanocapsules combined with immune checkpoint blockers in treating tumors

[0055] 40 mice were randomly divided into 4 groups, 10 in each group. 400,000 mouse skin cancer cells B16F10 were injected subcutaneously into each C57BL / 6J mouse on day 0. For the first group of mice, n(LOx) was injected intratumorally (0.5 mg / kg) into the mice on days 7 and 9 after the injection of mouse skin cancer cells. For the second group of mice, phosphate buffered solution of the same volume as the first group was injected intratumorally on days 7 and 9 after the injection of mouse skin cancer cells. For the third group of mice, aPD-L1 (100 μg / mouse) was injected intraperitoneally into the mice on days 9, 12, 16, and 19 after the injection of mouse skin cancer cells. For the fourth group of mice, n(LOx) was injected intratumorally (0.5 mg / kg) into the mice on the 7th and 9th days after the injection of mouse skin cancer cells, and aPD-L1 (100 μg / mouse) was injected intraperitoneally into the mice on the 9th, 12th, 16th and 19th days after the injection of mouse skin cancer cells.

[0056] 3 Experimental results

[0057] 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). Figure 1 As shown in the figure, compared with the lactate oxidase without nanoencapsulation, the lactate oxidase nanocapsules n(LOx) were retained in the sample wells, indicating that the volume of the nanoencapsulation was enlarged and it could not migrate quickly in the SDS-PEGE gel. Figure 2 As shown in the figure, the retention time of lactate oxidase nanocapsule n(LOx) in the chromatographic column is shorter than that of LOx, indicating that the molecular weight of LOx increases after nanoencapsulation, further proving the successful encapsulation of LOx. In addition, no UV absorption signal of LOx appears in the lactate oxidase nanocapsule n(LOx) sample, proving that the lactate oxidase nanocapsule n(LOx) is completely separated from lactate oxidase (LOx). Figure 1 The SDS-PAGE results were consistent with those of Figure 3 The results showed that the particle size of lactate oxidase nanocapsules n(LOx) was about 20nm and the Zeta potential was 1mV, which was significantly different from the lactate oxidase (LOx) without nanoencapsulation (7nm and -5.4mV). The above results showed that the monomer and cross-linker formed a cross-linked network on the surface of lactate oxidase to successfully encapsulate lactate oxidase.

[0058] To verify the effect of lactate oxidase in eliminating lactic acid and producing hydrogen peroxide, lactate oxidase nanocapsules n(LOx) were added to mouse breast cancer cells for co-incubation. The lactic acid concentration in the cell culture medium decreased rapidly, and hydrogen peroxide ( Figure 4 ). To verify the effect of lactate oxidase on immune cells, lactate oxidase nanocapsules n(LOx) were co-incubated with PBMCs in a 10mM lactic acid environment. After the addition of lactate oxidase nanocapsules n(LOx), the activation markers CD107a and CD69 of T cells were significantly increased ( Figure 5 ), interferon-γ (IFN-γ) is also secreted in large quantities ( Figure 6 ), proving that the addition of lactate oxidase nanocapsules n(LOx) can activate immune cells. 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 ). The above results show that lactate oxidase nanocapsules n(LOx) can eliminate lactic acid, produce hydrogen peroxide, activate immune cells, and especially activate and regulate T cells. Due to the regulatory function of lactate oxidase nanocapsules n(LOx) on the immune system, it has broad significance for the treatment of immune-related diseases caused by lactic acid accumulation.

[0059] The tumor inhibition effect of lactate oxidase nanocapsules (LOx) was further studied in a breast cancer mouse model. Figure 8 As shown, compared with the group injected with PBS only, both lactate oxidase (LOx) and lactate oxidase nanocapsules n (LOx) significantly inhibited tumor growth. Due to the protective effect of nanoencapsulation on lactate oxidase (LOx), lactate oxidase nanocapsules n (LOx) can more effectively inhibit tumor growth than unencapsulated lactate oxidase (LOx). In addition, the liver and kidney function indicators of mice were tested, and the results showed that lactate oxidase nanocapsules n (LOx) did not affect the liver and kidney function of mice ( Fig. 9 ), indicating that lactate oxidase nanocapsules n(LOx) have good biosafety and can be used as immune preparations in tumor immunotherapy.

[0060] In a skin cancer mouse model, the tumor inhibitory effect of lactate oxidase nanocapsules (LOx) combined with immune checkpoint blockers was further studied. Fig.10 As shown in Figure A, aPD-L1 itself is difficult to inhibit the growth of B16F10 tumors, and the tumors show basically the same exponential growth trend as the control group. n(LOx) effectively inhibits tumor growth, but the tumors still begin to grow about a week after the injection is stopped. In contrast, the combination of n(LOx) and aPD-L1 effectively inhibits tumor growth. Fig.10 The survival curve in Figure B also demonstrates that n(LOx) significantly improved the therapeutic effect of aPD-L1 in mouse skin cancer.

Claims

1. A lactate oxidase nanocapsule, characterized in that: The monomer and the cross-linking agent form a cross-linked network shell on the surface of the lactate oxidase, and the lactate oxidase is encapsulated in the shell.

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

3. The method for preparing a lactate oxidase nanocapsule according to claim 1 or 2, characterized in that: The preparation method is as follows: (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 in step (2) with a phosphate buffered saline solvent at room temperature; (4) At 4°C, a free radical polymerization reaction is initiated by an initiator on the surface of lactate oxidase, and the lactate oxidase is encapsulated in a network shell formed by a high molecular weight polymer to form lactate oxidase nanocapsules; (5) The obtained reaction product is dialyzed to obtain lactate oxidase nanocapsules n(LOx).

4. The method according to claim 3, characterized in that 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.

5. The method according to claim 3, characterized in that The crosslinking agent is one or more of N,N'-methylenebisacrylamide, glycerol dimethacrylate and triblock polylactic acid polyethylene glycol dimethacrylate.

6. The method according to claim 3, characterized in that The molar ratio of N-hydroxysuccinimide acrylate to lactate oxidase is 1:1-50. It is further preferred that the molar ratio of N-hydroxysuccinimide acrylate to lactate oxidase is 1:1-20; In the above scheme, 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.

7. The use of the lactate oxidase nanocapsules according to claim 1 or 2, wherein the lactate oxidase nanocapsules are used to prepare drugs that reduce the lactate concentration in tumors and release hydrogen peroxide in situ, or to prepare drugs that inhibit or treat tumors, or to prepare drugs for activating immune cells; or to prepare drugs for immunotherapy, or to prepare drugs for use in combination with tumor immunotherapy inhibitors for treating tumors.

8. According to the use of a lactate oxidase nanocapsule according to claim 7, 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).

9. The use according to claim 7 or 8, wherein the nanocapsules are prepared in the form of medicine for subcutaneous administration, intravenous administration and intratumoral injection.

10. A drug, characterized in that Comprising the lactate oxidase nanocapsules according to claim 1 or 2.

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