A weakly alkaline sodium salt nano drug, preparation method and application thereof
The weakly alkaline sodium salt nanodrug prepared by microemulsion method neutralizes lactic acid in the tumor microenvironment and induces pyrolysis, solving the problems of immunosuppression and pyrolysis inducers in tumor treatment, and achieving stronger anti-tumor Immune response and higher therapeutic effects.
Patent Information
- Application Number
- CN202310388724.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-12
- Publication Date
- 2024-12-17
- Estimated Expiration
- 2043-04-12
AI Technical Summary
Existing tumor treatment methods such as chemotherapy and radiotherapy have limited immune activation effects when inducing tumor cell apoptosis, and the blood circulation of pyroptosis inducers is fast and causing systemic adverse reactions. Single pyroptosis induces anti-tumor immune activation limited by immunosuppressed tumors. The microenvironment, and lactic acid inhibits the immune response in the tumor microenvironment.
The weakly alkaline sodium salt nanomedicine prepared by the microemulsion method consists of sodium oleate and ammonium bicarbonate. It can neutralize lactic acid in the tumor microenvironment, consume immunosuppressive factors, and reduce ions in tumor cells to induce cells. Activate immune response.
Through the synergistic effect of acid-base neutralization and cell pyroptosis, the systemic anti-tumor immune response is activated, the tumor treatment effect is improved, the immune suppression is reduced, the anti-tumor immune activity is enhanced, and the drug toxicity is reduced.
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Figure CN116395718B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedical nanomaterials, and in particular relates to a weakly alkaline sodium salt nanomedicine, a preparation method thereof and an application thereof. Background Art
[0002] The "Warburg effect" of tumors causes tumor cells to metabolize the large amount of glucose they take in to lactic acid through glycolysis even in the presence of sufficient oxygen. Lactic acid accumulated in the tumor microenvironment not only promotes tumor progression, but high concentrations of lactic acid are also transported into cells to be metabolized as a fuel substrate, while promoting tumor angiogenesis and invasion and metastasis. In addition, an increasing number of studies have shown that lactic acid in the tumor microenvironment can inhibit the body's immune response through various pathways, greatly affecting the effectiveness of tumor immunotherapy. For example, lactic acid derived from tumor cells can induce the expression of vascular endothelial growth factor and arginase 1 through the hypoxia-inducible factor 1-α signaling pathway, promote the polarization of tumor-associated macrophages to the M2 type, and promote tumor growth; lactic acid can inhibit the antigen presentation process and maturation and differentiation of dendritic cells; lactic acid can also inhibit the excretion of lactic acid produced by glycolysis of cytotoxic T cells, thereby inhibiting their cytotoxicity; at the same time, tumor-derived lactic acid can also reduce CD8 + The number and activity of T cells. Recent studies have also shown that tumor cells can not only avoid being killed by depriving effector T cells of nutrients, but also counteract the killing effect of effector T cells by supplying lactate to immunosuppressive regulatory T cells. In short, lactate, as a glycolytic metabolite, plays an important role in maintaining the immunosuppressive tumor microenvironment.
[0003] In addition, in traditional tumor treatment processes, such as classic chemotherapy and radiotherapy, cancer treatment is often achieved by inducing tumor cell apoptosis. However, cell apoptosis is generally considered to be non-immunogenic, so the in vivo immune activation effect of apoptosis-mediated tumor treatment is extremely limited. Cell pyroptosis is a type of programmed cell necrosis, which is a regulatable inflammatory cell death activated by Caspase and depends on members of the Gasdermins protein family to form cytoplasmic membrane pores. Recent studies have shown that cell pyroptosis can not only effectively kill tumor cells, but also the strong inflammatory response it triggers can efficiently induce the body to produce anti-tumor immune activity. However, common pyroptosis inducers such as small molecule chemotherapy drugs and active proteins are faced with problems such as rapid clearance from the blood circulation and systemic adverse reactions caused by high-dose drugs. Secondly, the anti-tumor immune activation induced by single pyroptosis is still greatly limited by the immunosuppressive tumor microenvironment, and the effect of immunotherapy needs to be further improved. Summary of the invention
[0004] In view of this, the purpose of the present invention is to provide a weakly alkaline sodium salt nanodrug, a preparation method and an application thereof, which can not only neutralize the acid-base with the lactic acid in the tumor microenvironment, consume the lactic acid and relieve immunosuppression; but also degrade in tumor cells to release a large amount of ions, induce cell pyroptosis and activate the immune response.
[0005] The invention provides a weakly alkaline sodium salt nano drug, which is prepared by a sodium oleate and ammonium bicarbonate microemulsion method.
[0006] In the present invention, the particle size of the weakly alkaline sodium salt nanomedicine is 10 to 200 nm.
[0007] The present invention provides a method for preparing the weakly alkaline sodium salt nanomedicine described in the above technical solution, comprising the following steps:
[0008] The ammonium bicarbonate and sodium oleate organic solution are mixed and reacted by a microemulsion method to obtain a weakly alkaline sodium salt nano drug.
[0009] In the present invention, the organic solvent in the sodium oleate organic solution is selected from a mixture of n-hexane, anhydrous ethanol and oleylamine.
[0010] In the present invention, the volume ratio of n-hexane, anhydrous ethanol and oleylamine in the mixture is (9-10):(2.5-3.5):1.
[0011] In the present invention, the ammonium bicarbonate is directly mixed with the sodium oleate organic solution;
[0012] Or dispersed in water and mixed with sodium oleate organic solution, the concentration of ammonium bicarbonate dispersed in water is 10-200 mg / mL. In a specific embodiment, the concentration of ammonium bicarbonate dispersed in water is 100 mg / mL, or 200 mg / mL, or 10 mg / mL.
[0013] In the present invention, the concentration of sodium oleate in the sodium oleate organic solution is 0.5-2.5 mg / mL. In a specific embodiment, the concentration of sodium oleate in the sodium oleate organic solution is 20 mg / 14 mL, or 0.5 mg / mL, or 35 mg / 14 mL.
[0014] In the present invention, the microemulsion reaction time is 3 to 60 minutes, and the microemulsion reaction temperature is 20 to 40° C. In a specific embodiment, the microemulsion reaction temperature is room temperature, and the reaction time is 3 minutes, 10 minutes, 30 minutes or 60 minutes.
[0015] In the present invention, ammonium bicarbonate and sodium oleate organic solution are mixed under stirring conditions of 200 to 1000 rpm. If ammonium bicarbonate is added in the form of an aqueous solution, the ammonium bicarbonate aqueous solution is preferably added dropwise to the sodium oleate organic solution.
[0016] The present invention provides a tumor treatment drug, comprising the weakly alkaline sodium salt nano drug described in the above technical solution or the weakly alkaline sodium salt nano drug prepared by the preparation method described in the above technical solution.
[0017] The present invention provides a weakly alkaline sodium salt nano drug, which is prepared by a sodium oleate and ammonium bicarbonate microemulsion method. The weakly alkaline sodium salt nano drug prepared by a sodium oleate and ammonium bicarbonate microemulsion method, namely NaHCO3, can not only neutralize the lactic acid in the tumor microenvironment, consume the lactic acid, and relieve the immunosuppressive tumor microenvironment; at the same time, it can degrade and release a large number of ions in tumor cells, induce cell pyroptosis, and activate immune response; thereby activating the systemic anti-tumor immune response through the synergistic effect of the two aspects to fight against tumor recurrence and metastasis and improve the therapeutic effect of tumors. The preparation method of the weakly alkaline sodium salt nano drug provided by the present invention is simple, the reaction conditions are mild, the raw material cost is low, and it has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is a transmission electron microscope photo of the weakly alkaline sodium salt nanomedicine obtained in Example 1 of the present invention;
[0019] Figure 2 The X-ray diffraction pattern of the weakly alkaline sodium salt nano drug obtained in Example 1 of the present invention;
[0020] Figure 3 This is a transmission electron microscope photo of the weakly alkaline sodium salt nanomedicine obtained in Example 9 of the present invention;
[0021] Figure 4 The pH value test result of the weakly alkaline sodium salt nano drug obtained in Example 1 of the present invention;
[0022] Figure 5 This is a photograph of the cell morphology of mouse breast cancer cells (4T1) treated with the weakly alkaline sodium salt nanomedicine obtained in Example 1 of the present invention;
[0023] Figure 6 The cytotoxicity data of the weakly alkaline sodium salt nano drug obtained in Example 1 of the present invention after treating 4T1 cells;
[0024] Figure 7 This is a transmission electron microscope photograph of the weakly alkaline sodium salt nanomedicine obtained in Example 8 of the present invention. DETAILED DESCRIPTION
[0025] In order to further illustrate the present invention, a weakly alkaline sodium salt nanomedicine provided by the present invention, its preparation method and application are described in detail below in combination with examples, but they should not be construed as limiting the scope of protection of the present invention.
[0026] Example 1
[0027] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 20 mg of ammonium bicarbonate was dispersed in 200 μl of deionized water. At room temperature, under the condition of a stirring rate of 1000 rpm, 200 μl of ammonium bicarbonate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature for 10 minutes. The precipitate was collected by centrifugation to obtain a weakly alkaline sodium salt nanomedicine.
[0028] The morphology of weakly alkaline sodium salt nanomedicine was characterized by transmission electron microscopy. Figure 1 As shown in the figure, it can be seen that the weakly alkaline sodium salt nanomedicine prepared by the microemulsion method is a spherical particle with uniform size. At the same time, X-ray diffraction was used to characterize the structure of the weakly alkaline sodium salt nanomedicine. The results are shown in Figure 2 As shown, it is proved that it has a pure phase structure of NaHCO3.
[0029] Example 2
[0030] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 20 mg of ammonium bicarbonate was dispersed in 100 μl of deionized water. At room temperature, under the condition of a stirring rate of 1000 rpm, 100 μl of ammonium bicarbonate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature for 10 minutes. The precipitate was collected by centrifugation to obtain a weakly alkaline sodium salt nano drug.
[0031] Example 3
[0032] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 10 mg of ammonium bicarbonate was dispersed in 1 ml of deionized water. At room temperature, under the condition of a stirring rate of 1000 rpm, 1 ml of ammonium bicarbonate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature for 10 minutes. The precipitate was collected by centrifugation to obtain a weakly alkaline sodium salt nano drug.
[0033] Example 4
[0034] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 20 mg of ammonium bicarbonate was dispersed in 200 μl of deionized water. At room temperature, under the condition of a stirring rate of 1000 rpm, 200 μl of ammonium bicarbonate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature for 3 minutes. The precipitate was collected by centrifugation to obtain a weakly alkaline sodium salt nanomedicine.
[0035] Example 5
[0036] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 20 mg of ammonium bicarbonate was dispersed in 200 μl of deionized water. At room temperature, under the condition of a stirring rate of 200 rpm, 200 μl of ammonium bicarbonate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature for 60 minutes. The precipitate was collected by centrifugation to obtain a weakly alkaline sodium salt nanomedicine.
[0037] Example 6
[0038] 7 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 20 mg of ammonium bicarbonate was dispersed in 200 μl of deionized water. At room temperature, under the condition of a stirring rate of 700 rpm, 200 μl of ammonium bicarbonate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature for 10 minutes. The precipitate was collected by centrifugation to obtain a weakly alkaline sodium salt nanomedicine.
[0039] Example 7
[0040] 35 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 20 mg of ammonium bicarbonate was dispersed in 200 μl of deionized water. At room temperature, under the condition of a stirring rate of 1000 rpm, 200 μl of ammonium bicarbonate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature for 10 minutes. The precipitate was collected by centrifugation to obtain a weakly alkaline sodium salt nanomedicine.
[0041] Example 8
[0042] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine. 20 mg of ammonium bicarbonate was added to the sodium oleate mixed solution at room temperature and a stirring rate of 1000 rpm, and the reaction was continued at room temperature for 10 minutes. The precipitate was collected by centrifugation to obtain a weakly alkaline sodium salt nano drug.
[0043] Example 9
[0044] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 30 mg of ammonium bicarbonate was dispersed in 300 μl of deionized water. At room temperature, 300 μl of ammonium bicarbonate aqueous solution was added dropwise to the sodium oleate mixed solution at a stirring rate of 1000 rpm, and the reaction was continued at room temperature for 30 minutes. The precipitate was collected by centrifugation to obtain a weakly alkaline sodium salt nanomedicine.
[0045] The present invention performs pH test on the weakly alkaline sodium salt nano drug obtained in Example 1. The test results are as follows: Figure 4 As shown, it is proved that the synthesized sodium salt nanomedicine is weakly alkaline.
[0046] The weakly alkaline sodium salt nanomedicine obtained in Example 1 was subjected to an in vitro cell pyroptosis detection experiment. The test method was as follows: 4T1 cells were seeded in a 12-well plate at a density of 100,000 cells per well. After culturing in RMPI medium for 12 hours, 250 micrograms per milliliter of sodium bicarbonate nanomedicine was added and incubated for 18 hours. The cell morphology was photographed using an inverted fluorescence microscope. The test results are shown in FIG. Figure 5 As shown, cells treated with weakly alkaline sodium salt nanomedicine showed obvious cell blebbing, proving the occurrence of cell pyroptosis.
[0047] The weakly alkaline sodium salt nanomedicine obtained in Example 1 was tested for in vitro cytotoxicity analysis. The test method is as follows: the cytotoxicity is determined by the MTT method. 4T1 cells are seeded in a 96-well plate at a density of 5000 cells per well. After culturing in RMPI medium for 12 hours, different concentrations of sodium bicarbonate nanomedicine (concentration range of 0-400 μg mL -1 ) were incubated for 24 hours, and cell proliferation was detected by MTT assay. Figure 6 As shown, Figure 6 The figure is a graph showing the inhibition of 4T1 cells by the sodium bicarbonate nanomedicine obtained in Example 1. Figure 6 It can be seen that the drug concentration is lower than 100 μg mL -1 There was no obvious toxicity to mouse cancer cells when the drug concentration reached 200 μg mL -1 When the drug concentration was increased to 400 μg·mL, the survival rate of 4T1 cells was only about 40%. -1 The survival rate of 4T1 cells was less than 10%. It can be concluded that sodium bicarbonate nanomedicine can achieve a certain inhibitory effect on 4T1 cells, and it is dose-dependent.
[0048] As can be seen from the above embodiments, the present invention provides a weakly alkaline sodium salt nano drug, which is prepared by a sodium oleate and ammonium bicarbonate microemulsion method. The weakly alkaline sodium salt nano drug prepared by a sodium oleate and ammonium bicarbonate microemulsion method, namely NaHCO3, can not only neutralize the lactic acid in the tumor microenvironment, consume lactic acid, and relieve the immunosuppressive tumor microenvironment; at the same time, it can degrade and release a large number of ions in tumor cells, induce cell pyroptosis, and activate immune response; thereby activating the systemic anti-tumor immune response through the synergistic effect of the two aspects to improve the therapeutic effect of tumors. The preparation method of the weakly alkaline sodium salt nano drug provided by the present invention is simple, the reaction conditions are mild, the raw material cost is low, and it has great application prospects.
[0049] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for preparing a weakly alkaline sodium salt nano drug, comprising the following steps: The ammonium bicarbonate and sodium oleate organic solution are mixed and reacted by a microemulsion method to obtain a weakly alkaline sodium salt nano drug; The particle size of the weakly alkaline sodium salt nano drug is 10 to 200 nm; The organic solvent in the sodium oleate organic solution is selected from a mixture of n-hexane, anhydrous ethanol and oleylamine; The volume ratio of n-hexane, anhydrous ethanol and oleylamine in the mixture is (9-10):(2.5-3.5):1; The concentration of sodium oleate in the sodium oleate organic solution is 0.5 to 2.5 mg / mL; Under stirring conditions of 200 to 1000 rpm, ammonium bicarbonate and sodium oleate organic solution are mixed; The reaction time of the microemulsion method is 3 to 60 minutes; the reaction temperature of the microemulsion method is 20 to 40°C.
2. The preparation method according to claim 1, characterized in that: The ammonium bicarbonate is directly mixed with the sodium oleate organic solution; Or dispersed in water and mixed with sodium oleate organic solution, the concentration of ammonium bicarbonate after being dispersed in water is 10-200 mg / mL.
3. A tumor treatment drug, characterized in that: The invention comprises a weakly alkaline sodium salt nano drug prepared by the preparation method according to any one of claims 1 to 2.