CN111569053A - Nano enzyme for enhancing immunotherapy by regulating and controlling tumor metabolic behaviors and preparation method and application thereof - Google Patents

一种通过调控肿瘤代谢行为增强免疫治疗的纳米酶及其制备 方法与应用A nanozyme for enhancing immunotherapy by regulating tumor metabolic behavior and its preparation methods and applications

技术领域technical field

本发明属于生物医用新材料领域，具体涉及一种通过调控肿瘤代谢行为增强免疫治疗的纳米酶及其制备方法与应用。The invention belongs to the field of new biomedical materials, in particular to a nanozyme for enhancing immunotherapy by regulating the metabolic behavior of tumors, and a preparation method and application thereof.

背景技术Background technique

癌症免疫疗法是通过调节人体的免疫系统，利用人体自身的免疫细胞去对抗肿瘤，并能够引发长期的抗肿瘤免疫响应。2018年，诺贝尔生理学或医学奖授予两位免疫学家，以表彰他们发现了抑制免疫负调节的癌症疗法。目前临床使用最多的免疫疗法是检查点疗法。但大多数肿瘤对免疫检查点疗法响应率较低(Yarchoan M,Hopkins A,Jaffee EM.Tumor mutational burden and response rate to PD-1inhibition[J].The NewEngland journal of medicine,2017,377(25):2500.)。影响治疗的最主要因素是免疫抑制微环境。因此解决肿瘤免疫微环境抑制的问题，可以使癌症免疫疗法发挥最大效果。Cancer immunotherapy modulates the body's immune system, uses the body's own immune cells to fight tumors, and can trigger a long-term anti-tumor immune response. In 2018, the Nobel Prize in Physiology or Medicine was awarded to two immunologists for discovering cancer therapies that suppress negative immune regulation. The most commonly used immunotherapy in clinical practice is checkpoint therapy. However, most tumors have a low response rate to immune checkpoint therapy (Yarchoan M, Hopkins A, Jaffee EM. Tumor mutational burden and response rate to PD-1 inhibition[J]. The NewEngland journal of medicine,2017,377(25): 2500.). The most important factor affecting treatment is the immunosuppressive microenvironment. Therefore, solving the problem of tumor immune microenvironment inhibition can maximize the effect of cancer immunotherapy.

代谢是生物体内所发生的用于维持生命的一系列有序的化学反应的总称。1927年，诺贝尔奖获得者Otto Warburg观察到肿瘤细胞消耗的葡萄糖是正常细胞的200倍，表现出显著不同的代谢表型；2010年，Craig B.Thompson发现了第一个肿瘤代谢产物，2-羟基戊二酸盐(参见Ward P S,Patel J,Wise D R,et al.The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity convertingα-ketoglutarate to 2-hydroxyglutarate[J].Cancer cell,2010,17(3):225-234.)。随着研究的深入，研究人员发现肿瘤细胞有五条代谢途径：有氧糖酵解、谷氨酰胺分解、一碳代谢、戊糖磷酸通路和脂肪酸从头合成。这五条代谢通路使肿瘤细胞由单纯的产生ATP转变为产生大量氨基酸、核苷酸、脂肪酸及细胞快速生长与增殖需要的其他中间产物。Metabolism is the general term for a series of ordered chemical reactions that take place in the body to sustain life. In 1927, Nobel laureate Otto Warburg observed that tumor cells consume 200 times more glucose than normal cells, showing significantly different metabolic phenotypes; in 2010, Craig B. Thompson discovered the first tumor metabolite, 2 -Hydroxyglutarate (see Ward P S, Patel J, Wise D R, et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting α-ketoglutarate to 2-hydroxyglutarate[J]. Cancer cell, 2010, 17(3):225-234.). With the deepening of the research, the researchers found that tumor cells have five metabolic pathways: aerobic glycolysis, glutaminolysis, one-carbon metabolism, pentose phosphate pathway and de novo synthesis of fatty acids. These five metabolic pathways transform tumor cells from simply producing ATP to producing a large amount of amino acids, nucleotides, fatty acids and other intermediates required for rapid cell growth and proliferation.

近年来，有研究证实肿瘤细胞的代谢异常，造成了肿瘤免疫抑制微环境，代谢失调会抑制免疫细胞浸润，影响细胞表面标志物的表达等，从而干扰免疫监测。目前已发现的与免疫疗法耐药性相关的代谢途径包括缺氧诱导因子、腺苷等。因此通过调控肿瘤代谢行为增强免疫治疗的研究将拥有巨大的发展潜力。In recent years, studies have confirmed that the abnormal metabolism of tumor cells creates a tumor immunosuppressive microenvironment. Metabolic disorders can inhibit the infiltration of immune cells and affect the expression of cell surface markers, thereby interfering with immune monitoring. Metabolic pathways that have been found to be related to immunotherapy resistance include hypoxia-inducible factor, adenosine, etc. Therefore, the research on enhancing immunotherapy by regulating tumor metabolic behavior will have great development potential.

发明内容SUMMARY OF THE INVENTION

有鉴于此，本发明的目的在于提供一种通过调控肿瘤代谢行为增强免疫治疗的纳米酶及其制备方法与应用。In view of this, the purpose of the present invention is to provide a nanozyme for enhancing immunotherapy by regulating the metabolic behavior of tumors, and a preparation method and application thereof.

为实现本发明的目的，本发明采用如下技术方案。To achieve the purpose of the present invention, the present invention adopts the following technical solutions.

一种纳米酶，由氧化酶、金属离子、2-甲基咪唑和透明质酸制成。A nanozyme made from oxidase, metal ions, 2-methylimidazole and hyaluronic acid.

在本发明中，所述纳米酶中所述氧化酶、金属离子与2-甲基咪唑的质量比为1：(0.1～50)：(0.01～30)。In the present invention, the mass ratio of the oxidase, the metal ion and the 2-methylimidazole in the nanozyme is 1:(0.1-50):(0.01-30).

在本发明中，所述纳米酶中所述氧化酶/金属离子/2-甲基咪唑与透明质酸的质量比为1：(0.01～10)。In the present invention, the mass ratio of the oxidase/metal ion/2-methylimidazole to hyaluronic acid in the nanozyme is 1:(0.01-10).

在本发明中，所述纳米酶中所述氧化酶为葡萄糖氧化酶和乳酸氧化酶中的一种或两种；所述金属离子为Mn²⁺和Cu²⁺中的一种或两种。In the present invention, the oxidase in the nanozyme is one or both of glucose oxidase and lactate oxidase; the metal ion is one or both of Mn ²⁺ and Cu ²⁺ .

在本发明中，所述纳米酶中所述葡萄糖氧化酶的分子量为100000～160000Da；所述乳酸氧化酶的分子量为300000～350000Da。In the present invention, the molecular weight of the glucose oxidase in the nanozyme is 100,000-160,000 Da; the molecular weight of the lactate oxidase is 300,000-350,000 Da.

在本发明中，所述纳米酶中所述Mn²⁺为四水合氯化锰，所述Cu²⁺为二水合氯化铜。In the present invention, the Mn ²⁺ in the nanozyme is manganese chloride tetrahydrate, and the Cu ²⁺ is copper chloride dihydrate.

本发明还提供了上述纳米酶的制备方法，其特征在于，包括如下步骤：The present invention also provides the preparation method of the above-mentioned nano-enzyme, which is characterized in that, it comprises the following steps:

A、将氧化酶与2-甲基咪唑溶于水先进行作用，然后加入金属离子混合，离心弃上清，超声分散后，制得氧化酶/金属离子/2-甲基咪唑；A. Dissolve the oxidase and 2-methylimidazole in water to act first, then add metal ions to mix, centrifuge to discard the supernatant, and ultrasonically disperse to obtain the oxidase/metal ion/2-methylimidazole;

B、将透明质酸水溶液与氧化酶/金属离子/2-甲基咪唑混匀，静止复合得到所述纳米酶。B. Mix the hyaluronic acid aqueous solution with oxidase/metal ion/2-methylimidazole, and statically compound to obtain the nanozyme.

本发明所述制备方法首先将氧化酶与2-甲基咪唑先进行作用，然后加入金属离子与2-甲基咪唑进行配位共混，实现原位载氧化酶，最后用透明质酸修饰，得到氧化酶/金属离子/2-甲基咪唑/透明质酸纳米酶。The preparation method of the invention firstly reacts the oxidase with 2-methylimidazole, then adds metal ions and 2-methylimidazole for coordination and blending to realize the in-situ carrying of the oxidase, and finally modifies with hyaluronic acid, Oxidase/metal ion/2-methylimidazole/hyaluronic acid nanozyme was obtained.

在本发明中，所述的制备方法中步骤A中氧化酶溶于水的浓度为1.5mg/mL。In the present invention, the concentration of the oxidase dissolved in water in step A of the preparation method is 1.5 mg/mL.

在本发明中，所述的制备方法中步骤A所述氧化酶与2-甲基咪唑作用时间为10～60min。在一些实施方案中，所述作用时间为20～40min。In the present invention, in the preparation method, the reaction time of the oxidase in step A with 2-methylimidazole is 10-60 min. In some embodiments, the action time is 20-40 min.

在本发明中，所述的制备方法中步骤A所述加入金属离子0.5～2h后进行离心。在一些实施方案中，所述加入金属离子1～1.5h后进行离心。In the present invention, in the preparation method described in step A, the metal ions are added for 0.5 to 2 hours and then centrifuged. In some embodiments, centrifugation is performed 1-1.5 hours after the addition of metal ions.

进一步的，所述离心速率为8000～12000rpm，所述离心时间为10～30min。在一些实施方案中，所述离心速率为10000～12000rpm，时间为20～30min。Further, the centrifugal speed is 8000-12000 rpm, and the centrifugal time is 10-30 min. In some embodiments, the centrifugation rate is 10000-12000 rpm for 20-30 min.

在本发明中，所述的制备方法中步骤A所述超声分散时间为0.5～15min。在一些实施方案中，所述超声分散时间为0.5～5min。In the present invention, in the preparation method, the ultrasonic dispersion time in step A is 0.5-15 min. In some embodiments, the ultrasonic dispersion time is 0.5-5 min.

在本发明中，所述的制备方法中步骤B中所述透明质酸水溶液与氧化酶/金属离子/2-甲基咪唑等体积。In the present invention, in the preparation method, the hyaluronic acid aqueous solution and the oxidase/metal ion/2-methylimidazole in step B are equal in volume.

在本发明中，所述的制备方法中步骤B所述静置时间为10～60min。在一些实施方案中，所述静置时间为20～30min。In the present invention, in the preparation method, the standing time in step B is 10-60 min. In some embodiments, the resting time is 20-30 min.

本发明所述纳米酶经尾静脉给药后到达肿瘤区域内释放氧化酶，通过饥饿疗法调控肿瘤代谢行为；其次，金属离子可以与过氧化氢发生类芬顿反应产生活性氧，杀伤肿瘤。因此本发明还提供了所述的纳米酶在制备调控肿瘤代谢、杀伤肿瘤的药物中的应用。The nanozyme of the present invention reaches the tumor area to release oxidase after being administered through the tail vein, and regulates tumor metabolism through starvation therapy; secondly, metal ions can undergo Fenton-like reaction with hydrogen peroxide to generate reactive oxygen species to kill tumors. Therefore, the present invention also provides the application of the nanozyme in the preparation of drugs for regulating tumor metabolism and killing tumors.

此外，本发明所述纳米酶可以通过调控肿瘤代谢行为增强免疫治疗，增敏PD-L1抗体治疗，从而实现饥饿治疗、化学动力学治疗和免疫治疗相联合，达到更好的治疗效果。因此本发明还提供了所述的纳米酶在制备PD-L1抗体增敏剂中的应用。In addition, the nanozyme of the present invention can enhance immunotherapy and sensitize PD-L1 antibody therapy by regulating tumor metabolic behavior, so as to realize the combination of starvation therapy, chemodynamic therapy and immunotherapy to achieve better therapeutic effect. Therefore, the present invention also provides the application of the nanozyme in the preparation of PD-L1 antibody sensitizers.

由上述技术方案可知，本发明提供了一种纳米酶及其制备方法与应用。本发明所述纳米酶由氧化酶、金属离子、2-甲基咪唑和透明质酸制成，可以通过调控肿瘤代谢行为增强免疫治疗，增敏PD-L1抗体治疗，从而实现饥饿治疗、化学动力学治疗和免疫治疗相联合，达到更好的治疗效果。本发明所述纳米酶的制备方法具有易于操作、价格低廉等优点。As can be seen from the above technical solutions, the present invention provides a nanozyme and a preparation method and application thereof. The nanozyme of the present invention is made of oxidase, metal ions, 2-methylimidazole and hyaluronic acid, and can enhance immunotherapy and sensitize PD-L1 antibody treatment by regulating tumor metabolic behavior, thereby realizing starvation treatment, chemical kinetics Combination of chemotherapy and immunotherapy to achieve better therapeutic effect. The preparation method of the nanozyme of the present invention has the advantages of easy operation, low price and the like.

附图说明Description of drawings

为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.

图1为本发明实施例3中得到的葡萄糖氧化酶/锰离子/2-甲基咪唑的扫描电镜图；Fig. 1 is the scanning electron microscope picture of glucose oxidase/manganese ion/2-methylimidazole obtained in the embodiment of the present invention 3;

图2为本发明实施例20中得到的纳米酶的细胞毒性；Figure 2 is the cytotoxicity of the nanozyme obtained in Example 20 of the present invention;

图3为本发明实施例28中得到的细胞水平消耗葡萄糖能力图；(1)PBS组，(2)葡萄糖氧化酶组，(3)锰离子/2-甲基咪唑/透明质酸组，(4)实施例20得到的纳米酶组；Figure 3 is a graph of the glucose consumption ability at the cellular level obtained in Example 28 of the present invention; (1) PBS group, (2) glucose oxidase group, (3) manganese ion/2-methylimidazole/hyaluronic acid group, ( 4) Nanozyme group obtained in Example 20;

图4为本发明实施例29中得到的细胞水平活性氧染色图；(1)PBS组，(2)葡萄糖氧化酶组，(3)锰离子/2-甲基咪唑/透明质酸+过氧化氢组，(4)实施例20得到的纳米酶组；Fig. 4 is the cell-level reactive oxygen species staining diagram obtained in Example 29 of the present invention; (1) PBS group, (2) glucose oxidase group, (3) manganese ion/2-methylimidazole/hyaluronic acid+peroxidation Hydrogen group, (4) Nanozyme group obtained in Example 20;

图5为本发明实施例30中得到的流式细胞术对肿瘤细胞上的PD-L1表达检测结果图；(1)PBS组，(2)锰离子/2-甲基咪唑组，(3)葡萄糖氧化酶/锰离子/2-甲基咪唑组，(4)实施例20得到的纳米酶组，(5)PD-L1抗体组，(6)实施例20得到的纳米酶+PD-L1抗体组。Fig. 5 is a graph showing the detection results of PD-L1 expression on tumor cells by flow cytometry obtained in Example 30 of the present invention; (1) PBS group, (2) manganese ion/2-methylimidazole group, (3) Glucose oxidase/manganese ion/2-methylimidazole group, (4) Nanozyme group obtained in Example 20, (5) PD-L1 antibody group, (6) Nanozyme+PD-L1 antibody obtained in Example 20 Group.

具体实施方式Detailed ways

本发明公开了一种通过调控肿瘤代谢行为增强免疫治疗的纳米酶及其制备方法与应用。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及产品已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法进行改动或适当变更与组合，来实现和应用本发明技术。The invention discloses a nanozyme for enhancing immunotherapy by regulating the metabolic behavior of tumors, and a preparation method and application thereof. Those skilled in the art can learn from the content of this document and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and product of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention. Invention technology.

根据本发明，所述纳米酶，由氧化酶、金属离子、2-甲基咪唑和透明质酸组成。首先将氧化酶与2-甲基咪唑先进行作用，然后加入金属离子与2-甲基咪唑进行配位共混，实现原位载氧化酶，最后用透明质酸修饰，制备得到所述纳米酶。According to the present invention, the nanozyme is composed of oxidase, metal ion, 2-methylimidazole and hyaluronic acid. First, the oxidase and 2-methylimidazole are reacted first, and then metal ions are added to coordinate and blend with 2-methylimidazole to realize in-situ loading of oxidase. Finally, the nanozyme is prepared by modifying it with hyaluronic acid. .

根据本发明，所述葡萄糖氧化酶的分子量为100000～160000Da。在一些实施方案中为140000～160000Da。在一些实施例中为150000Da。According to the present invention, the molecular weight of the glucose oxidase is 100000-160000 Da. In some embodiments from 140,000 to 160,000 Da. In some embodiments it is 150000 Da.

根据本发明，所述乳酸氧化酶的分子量为300000～350000Da。在一些实施方案中为330000～350000Da。在一些实施例中为350000Da。According to the present invention, the molecular weight of the lactate oxidase is 300,000-350,000 Da. In some embodiments from 330,000 to 350,000 Da. In some embodiments it is 350000 Da.

所述氧化酶、金属离子与2-甲基咪唑的比例为1：(0.1～50)：(0.01～30)。在一些实施方案中为1：(0.5～20)：(0.1～10)。进一步的，在一些实施例中为1:(1～10)：(0.6～4)。The ratio of the oxidase, metal ion and 2-methylimidazole is 1:(0.1-50):(0.01-30). In some embodiments it is 1:(0.5-20):(0.1-10). Further, in some embodiments, it is 1:(1-10):(0.6-4).

所述氧化酶/金属离子/2-甲基咪唑与透明质酸的比例为1：(0.01～10)，在一些实施方案中为1：(0.01～3)。The ratio of the oxidase/metal ion/2-methylimidazole to hyaluronic acid is 1:(0.01-10), in some embodiments, 1:(0.01-3).

按照本发明，所述纳米酶的制备方法具体如下：氧化酶与2-甲基咪唑均溶于水中，先进行作用，然后加入金属离子水溶液与2-甲基咪唑进行配位，离心。弃上清，超声分散后，制备出氧化酶/金属离子/2-甲基咪唑。再用透明质酸水溶液与氧化酶/金属离子/2-甲基咪唑混匀，静止复合，得到氧化酶/金属离子/2-甲基咪唑/透明质酸纳米酶。According to the present invention, the specific preparation method of the nanozyme is as follows: both the oxidase and 2-methylimidazole are dissolved in water, and they act firstly, and then an aqueous solution of metal ions is added to coordinate with 2-methylimidazole, and then centrifuged. The supernatant was discarded, and after ultrasonic dispersion, the oxidase/metal ion/2-methylimidazole was prepared. The hyaluronic acid aqueous solution is then mixed with oxidase/metal ion/2-methylimidazole, and statically compounded to obtain oxidase/metal ion/2-methylimidazole/hyaluronic acid nanozyme.

根据本发明，所述氧化酶与2-甲基咪唑作用时间为10～60min，在一些实施方案中为20～40min。According to the present invention, the reaction time of the oxidase and 2-methylimidazole is 10-60 min, in some embodiments, 20-40 min.

所述加入金属离子与2-甲基咪唑配位作用时间为0.5～2h，在一些实施方案中为1～1.5h。The time for the addition of metal ions to coordinate with 2-methylimidazole is 0.5-2 h, and in some embodiments, 1-1.5 h.

所述离心速率为8000～12000rpm，在一些实施方案中为10000～12000rpm；所述离心时间为10～30min，在一些实施方案中为20～30min。The centrifugation rate is 8000-12000 rpm, in some embodiments, 10,000-12,000 rpm; the centrifugation time is 10-30 min, and in some embodiments, 20-30 min.

所述超声分散时间为0.5～15min，在一些实施方案中为0.5～5min。The ultrasonic dispersion time is 0.5-15 min, in some embodiments 0.5-5 min.

所述静置时间为10～60min，在一些实施方案中为20～30min。The resting time is 10-60 min, in some embodiments 20-30 min.

根据本发明，所述通过调控肿瘤代谢行为增强免疫治疗的纳米酶经尾静脉给药后到达肿瘤区域内释放氧化酶，通过饥饿疗法调控肿瘤代谢行为；其次，金属离子可以与过氧化氢发生类芬顿反应产生活性氧，杀伤肿瘤；此外，该纳米酶可以通过调控肿瘤代谢行为增强免疫治疗，增敏PD-L1抗体治疗，从而实现饥饿治疗、化学动力学治疗和免疫治疗相联合，达到更好的治疗效果。According to the present invention, the nanozyme that enhances immunotherapy by regulating tumor metabolic behavior is administered through the tail vein and reaches the tumor area to release oxidase, and the tumor metabolic behavior is regulated by starvation therapy; secondly, metal ions can interact with hydrogen peroxide. The Fenton reaction generates reactive oxygen species and kills tumors; in addition, the nanozyme can enhance immunotherapy and sensitize PD-L1 antibody treatment by regulating tumor metabolic behavior, so as to realize the combination of starvation therapy, chemodynamic therapy and immunotherapy to achieve better results. good therapeutic effect.

本发明所述各实施例涉及的试验方按照以下操作进行：The test party involved in each embodiment of the present invention is carried out according to the following operations:

细胞培养：在本发明中，选用4T1细胞系，所述细胞培养方法按照通用方法，无特殊限制。培养基优选为含10％胎牛血清的DMEM培养基，所述培养条件优选在二氧化碳体积分数为5％，温度为37℃的培养箱中。Cell culture: In the present invention, the 4T1 cell line is selected, and the cell culture method is based on a general method without special restrictions. The medium is preferably DMEM medium containing 10% fetal bovine serum, and the culture conditions are preferably in an incubator with a carbon dioxide volume fraction of 5% and a temperature of 37°C.

细胞毒性：细胞毒性评价选用4T1细胞系。将细胞按照每孔8×10³的密度种于96孔板中，培养过夜。不同浓度的材料与细胞共培养24h后，每孔加入20μL的CCK-8溶液，继续培养1h。通过酶标仪检测每孔在450nm和610nm下吸光度值。并通过以下公式计算细胞存活率。Cytotoxicity: 4T1 cell line was selected for cytotoxicity evaluation. Cells were seeded in 96-well plates at a density of 8×10 ³ per well and cultured overnight. After co-culturing the cells with different concentrations of materials for 24 hours, 20 μL of CCK-8 solution was added to each well, and the culture was continued for 1 hour. The absorbance values of each well at 450 nm and 610 nm were detected by a microplate reader. And the cell viability was calculated by the following formula.

细胞存活率(％)＝(A_样品/A_空白)×100Cell viability (%)=(A _sample /A _blank )×100

细胞水平消耗葡萄糖能力：细胞水平消耗葡萄糖能力评价选用4T1细胞系。将细胞按照每孔1×10⁵的密度种于24孔板中，培养过夜。将不同的材料与细胞共培养24h后，用血糖仪测定各孔培养基中糖含量。Cell-level glucose consumption ability: 4T1 cell line was selected for the evaluation of cell-level glucose consumption ability. Cells were seeded in 24-well plates at a density of 1×10 ⁵ per well and cultured overnight. After co-culturing the cells with different materials for 24 hours, the sugar content in the culture medium of each well was measured with a blood glucose meter.

细胞水平活性氧染色：细胞水平活性氧染色评价选用4T1细胞系。将细胞按照每孔1×10⁵的密度种于24孔板中，培养过夜。将不同的材料与细胞共培养。按照1:1000用无血清培养液稀释2',7'-二氯荧光黄双乙酸盐，使终浓度为10μmol/L。去除细胞培养液，加入适当体积稀释好的2',7'-二氯荧光黄双乙酸盐，37℃细胞培养箱内孵育20min，用无血清的培养液洗涤细胞三次，使用488nm激发波长，525nm发射波长，用荧光显微镜检测。Cell-level reactive oxygen species staining: The 4T1 cell line was selected for the evaluation of cell-level reactive oxygen species staining. Cells were seeded in 24-well plates at a density of 1×10 ⁵ per well and cultured overnight. The different materials were co-cultured with the cells. 2',7'-Dichlorofluorescein diacetate was diluted 1:1000 with serum-free medium to a final concentration of 10 μmol/L. Remove the cell culture medium, add an appropriate volume of diluted 2',7'-dichlorofluorescein diacetate, incubate in a 37°C cell incubator for 20 minutes, wash the cells three times with serum-free medium, use 488nm excitation wavelength, 525nm emission wavelength, detected with a fluorescence microscope.

联合治疗动物模型：选用4T1肿瘤模型，并选用20g左右的Balb/C小白鼠。在小鼠右后肢外侧皮下接种密度为1×10⁶的4T1细胞，待瘤体积长至100mm³左右时，将通过调控肿瘤代谢行为增强免疫治疗的纳米酶尾静脉注射到小鼠体内，共进行4次给药，联合治疗组再进行2次PD-L1抗体腹腔给药。跟踪瘤体积大小，实验结束后通过流式细胞术对肿瘤细胞上的PD-L1表达进行检测。Animal model for combined therapy: 4T1 tumor model and Balb/C mice of about 20 g were selected. 4T1 cells at a density of 1×10 ⁶ were subcutaneously inoculated on the lateral side of the right hind limb of the mice. When the tumor volume grew to about 100 mm ³ , the nanozyme, which can enhance immunotherapy by regulating tumor metabolism, was injected into the mice through the tail vein. 4 times of administration, the combination treatment group received 2 more times of intraperitoneal administration of PD-L1 antibody. The tumor volume was tracked, and the PD-L1 expression on tumor cells was detected by flow cytometry after the experiment.

为了进一步理解本发明，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. . Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, which can be purchased through commercial channels.

实施例1～16Examples 1 to 16

首先氧化酶与2-甲基咪唑均溶于水中，先进行作用30min，然后加入金属离子水溶液与2-甲基咪唑进行配位1h后离心，离心转速为12000rpm，时间为20min。弃上清，超声分散30s后，制备出氧化酶/金属离子/2-甲基咪唑。其中，氧化酶、金属离子与2-甲基咪唑的用量见表1。First, both oxidase and 2-methylimidazole were dissolved in water, and the reaction was performed for 30 minutes. Then, an aqueous solution of metal ions was added to coordinate with 2-methylimidazole for 1 hour, and then centrifuged at 12,000 rpm for 20 minutes. The supernatant was discarded, and oxidase/metal ion/2-methylimidazole was prepared after ultrasonic dispersion for 30s. Wherein, the consumption of oxidase, metal ion and 2-methylimidazole is shown in Table 1.

表1实施例1～16不同原料的用量The consumption of table 1 embodiment 1～16 different raw materials

按照实施例3用量比例，制备出葡萄糖氧化酶/锰离子/2-甲基咪唑，利用扫描电镜对实施例3中得到的葡萄糖氧化酶/锰离子/2-甲基咪唑进行分析，得到扫描电镜图片，如图1所示。结果显示，实施例3制备出的葡萄糖氧化酶/锰离子/2-甲基咪唑平均大小在40～80nm之间。According to the dosage ratio of Example 3, glucose oxidase/manganese ion/2-methylimidazole was prepared, and the glucose oxidase/manganese ion/2-methylimidazole obtained in Example 3 was analyzed by scanning electron microscope to obtain a scanning electron microscope picture, as shown in Figure 1. The results showed that the average size of the glucose oxidase/manganese ion/2-methylimidazole prepared in Example 3 was between 40 and 80 nm.

实施例1-2、5-6粒径比实施例3小，实施例4、7-16粒径比实施例3大。The particle sizes of Examples 1-2 and 5-6 are smaller than those of Example 3, and the particle sizes of Examples 4 and 7-16 are larger than those of Example 3.

实施例17～26Examples 17 to 26

按照实施例3，制备出的葡萄糖氧化酶/锰离子/2-甲基咪唑，再用等体积的透明质酸水溶液与葡萄糖氧化酶/锰离子/2-甲基咪唑混匀，静止复合20min，得到葡萄糖氧化酶/锰离子/2-甲基咪唑/透明质酸纳米酶。其中，葡萄糖氧化酶/锰离子/2-甲基咪唑与透明质酸的用量见表2。According to Example 3, the prepared glucose oxidase/manganese ion/2-methylimidazole was mixed with an equal volume of hyaluronic acid aqueous solution and glucose oxidase/manganese ion/2-methylimidazole, and the mixture was static for 20min. Glucose oxidase/manganese ion/2-methylimidazole/hyaluronic acid nanozyme was obtained. Wherein, the consumption of glucose oxidase/manganese ion/2-methylimidazole and hyaluronic acid is shown in Table 2.

表2实施例17～26不同原料的用量The consumption of table 2 embodiment 17～26 different raw materials

按照上表用量制备出各实施例17-26的纳米酶。The nanozymes of Examples 17-26 were prepared according to the dosage in the above table.

实施例27Example 27

将4T1细胞按照每孔8×10³的密度种于96孔板中，培养过夜。不同浓度的实施例20得到的纳米酶与细胞共培养24h后，每孔加入20μL的CCK-8溶液，继续培养1h。通过酶标仪检测每孔在450nm和610nm下吸光度值。计算细胞存活率，结果如图2所示。4T1 cells were seeded in 96-well plates at a density of 8×10 ³ per well and cultured overnight. After the nanozymes obtained in Example 20 with different concentrations were co-cultured with cells for 24 hours, 20 μL of CCK-8 solution was added to each well, and the culture was continued for 1 hour. The absorbance values of each well at 450 nm and 610 nm were detected by a microplate reader. The cell viability was calculated and the results are shown in Figure 2.

实验结果表明，实施例20得到的纳米酶在较低浓度时就对4T1细胞具有杀伤作用。The experimental results show that the nanozyme obtained in Example 20 has a killing effect on 4T1 cells at a lower concentration.

实施例17-19、21-26得到的纳米酶对4T1细胞的杀伤作用与实施例20相似。The killing effects of the nanozymes obtained in Examples 17-19 and 21-26 on 4T1 cells were similar to those in Example 20.

实施例28Example 28

将4T1细胞按照每孔1×10⁵的密度接种于24孔板中，培养过夜。实验分为四组：(1)PBS组，(2)葡萄糖氧化酶组，(3)锰离子/2-甲基咪唑/透明质酸组，(4)实施例20得到的纳米酶组，分别与细胞共培养，(2)组和(4)组按葡萄糖氧化酶终浓度为2μg/mL加入，(1)组加入与其他组等体积的PBS，(3)组的用量按锰离子/2-甲基咪唑/透明质酸计保持与(4)组中的锰离子/2-甲基咪唑/透明质酸一致，24h后用血糖仪测定各孔培养基中糖含量，结果如图3所示。4T1 cells were seeded in 24-well plates at a density of 1×10 ⁵ per well and cultured overnight. The experiment was divided into four groups: (1) PBS group, (2) glucose oxidase group, (3) manganese ion/2-methylimidazole/hyaluronic acid group, (4) nanozyme group obtained in Example 20, respectively. Co-cultured with cells, groups (2) and (4) were added at a final concentration of glucose oxidase of 2 μg/mL, group (1) was added with the same volume of PBS as other groups, and the dosage of group (3) was manganese ion/2 -The methylimidazole/hyaluronic acid meter was kept consistent with the manganese ion/2-methylimidazole/hyaluronic acid in group (4). After 24 hours, the sugar content in the culture medium of each well was measured with a blood glucose meter. The results are shown in Figure 3. Show.

实验结果表明，相对于(1)组和(3)组，用(4)组处理后，培养基中葡萄糖含量下降，且下降量与(2)组相差不多。所以实施例20得到的纳米酶具有消耗葡萄糖的能力。The experimental results showed that, compared with groups (1) and (3), after treatment with group (4), the glucose content in the medium decreased, and the amount of decrease was similar to that of group (2). Therefore, the nanozyme obtained in Example 20 has the ability to consume glucose.

实施例17-19、21-26得到的纳米酶消耗葡萄糖的能力与实施例20相似。The ability of the nanozymes obtained in Examples 17-19 and 21-26 to consume glucose was similar to that of Example 20.

实施例29Example 29

将4T1细胞按照每孔1×10⁵的密度种于24孔板中，培养过夜。实验分为四组：(1)PBS组，(2)葡萄糖氧化酶组，(3)锰离子/2-甲基咪唑/透明质酸+过氧化氢组，(4)实施例20得到的纳米酶组，分别与细胞共培养，(2)组和(4)组按葡萄糖氧化酶终浓度为5μg/mL加入，(1)组加入与其他组等体积的PBS，(3)组的用量按锰离子/2-甲基咪唑/透明质酸计保持与(4)组中的锰离子/2-甲基咪唑/透明质酸用量一致，(3)组中的过氧化氢用量为1×10^-3μmol/L。按照1：1000用无血清培养液稀释2',7'-二氯荧光黄双乙酸盐，使终浓度为10μmol/L。去除细胞培养液，加入适当体积稀释好的2',7'-二氯荧光黄双乙酸盐，37℃细胞培养箱内孵育20min，用无血清的培养液洗涤细胞三次，使用488nm激发波长，525nm发射波长，用荧光显微镜检测，结果如图4所示。4T1 cells were seeded in 24-well plates at a density of 1×10 ⁵ per well and cultured overnight. The experiment is divided into four groups: (1) PBS group, (2) glucose oxidase group, (3) manganese ion/2-methylimidazole/hyaluronic acid+hydrogen peroxide group, (4) nanometer obtained in Example 20 The enzyme group was co-cultured with cells, respectively. Groups (2) and (4) were added at a final concentration of glucose oxidase of 5 μg/mL. Group (1) was added with the same volume of PBS as the other groups. The amount of manganese ion/2-methylimidazole/hyaluronic acid is the same as the amount of manganese ion/2-methylimidazole/hyaluronic acid in group (4), and the amount of hydrogen peroxide in group (3) is 1×10 ^-3 μmol/L. Dilute 2',7'-dichlorofluorescein diacetate with serum-free medium at 1:1000 to make the final concentration 10 μmol/L. Remove the cell culture medium, add an appropriate volume of diluted 2',7'-dichlorofluorescein diacetate, incubate in a 37°C cell incubator for 20 minutes, wash the cells three times with serum-free medium, use 488nm excitation wavelength, 525nm emission The wavelengths were detected with a fluorescence microscope, and the results are shown in Figure 4.

实验结果表明，(1)组和(2)组未产生绿色荧光，即未有活性氧产生，而(3)组和(4)组，产生大量绿色荧光，即有大量活性氧产生。表明实施例20得到的纳米酶中Mn²⁺可以与葡萄糖氧化酶消耗葡萄糖产生的过氧化氢发生类芬顿反应产生活性氧，杀伤肿瘤。The experimental results showed that groups (1) and (2) did not produce green fluorescence, that is, no reactive oxygen species were produced, while groups (3) and (4) produced a large amount of green fluorescence, that is, a large amount of reactive oxygen species was produced. It is shown that Mn ²⁺ in the nanozyme obtained in Example 20 can undergo a Fenton-like reaction with hydrogen peroxide generated by glucose oxidase consumption of glucose to generate reactive oxygen species and kill tumors.

实施例17-19、21-26得到的纳米酶与实施例20相似，也可以与葡萄糖氧化酶消耗葡萄糖产生的过氧化氢发生类芬顿反应产生活性氧，杀伤肿瘤。The nanozymes obtained in Examples 17-19 and 21-26 are similar to those in Example 20, and can also undergo a Fenton-like reaction with hydrogen peroxide generated by glucose oxidase consumption of glucose to generate reactive oxygen species and kill tumors.

实施例30Example 30

选用20g左右的Balb/C小白鼠，共分六组：(1)PBS组，(2)锰离子/2-甲基咪唑组，(3)葡萄糖氧化酶/锰离子/2-甲基咪唑组，(4)实施例20得到的纳米酶组，(5)PD-L1抗体组和(6)实施例20得到的纳米酶+PD-L1抗体组，每组5只。在小鼠右后肢外侧皮下接种密度为1×10⁶的4T1细胞，待瘤体积长至100mm³左右时，将各组尾静脉注射到小鼠体内，共进行4次给药，每次间隔2天(若第一次给药记为D0，则后三次分别为D3，D6，D9)，各组给药量按葡萄糖氧化酶终浓度为20μg/只，(2)组用量保持与(4)组中锰离子/2-甲基咪唑用量一致，(3)组用量与(4)中葡萄糖氧化酶/锰离子/2-甲基咪唑用量一致，(5)组与(6)组进行2次PD-L1抗体腹腔给药(分别为D4，D8)，给药量为50μg/只。跟踪瘤体积大小，开始治疗14天后实验结束，通过流式细胞术对肿瘤细胞上的PD-L1表达进行检测。结果见表3及图5。Balb/C mice of about 20g were selected and divided into six groups: (1) PBS group, (2) manganese ion/2-methylimidazole group, (3) glucose oxidase/manganese ion/2-methylimidazole group , (4) the nanozyme group obtained in Example 20, (5) the PD-L1 antibody group and (6) the nanozyme+PD-L1 antibody group obtained in Example 20, 5 in each group. 4T1 cells at a density of 1×10 ⁶ were subcutaneously inoculated on the lateral side of the right hind limb of the mice. When the tumor volume grew to about 100 mm ³ , the mice in each group were injected into the mice through the tail vein. Days (if the first administration is recorded as D0, then the next three times are respectively D3, D6, D9), the final dose of glucose oxidase in each group is 20 μg/dose, the dosage of (2) group remains the same as that of (4) The dosage of manganese ion/2-methylimidazole in the group was the same, the dosage of group (3) was the same as the dosage of glucose oxidase/manganese ion/2-methylimidazole in group (4), the dosage of group (5) and group (6) were carried out twice PD-L1 antibody was administered intraperitoneally (D4, D8, respectively), and the dose was 50 μg/only. The tumor volume was tracked, and the experiment ended 14 days after the start of treatment, and the PD-L1 expression on tumor cells was detected by flow cytometry. The results are shown in Table 3 and Figure 5.

表3各组肿瘤体积Table 3 Tumor volume in each group

分组grouping 肿瘤体积tumor volume (1)组(Group 1 2000mm³2000mm³ (2)组(2 teams 1500mm³1500mm³ (3)组(3) Group 1000mm³1000mm³ (4)组(4) Group 500mm³500mm³ (5)组(5) Group 1800mm³1800mm³ (6)组(6) Group 100mm³100mm³

实验结果表明，(6)组与其他对照组相比，完全抑制了肿瘤生长，所以饥饿治疗、化学动力学治疗和免疫治疗相联合可以达到更好的治疗效果。The experimental results showed that compared with other control groups, group (6) completely inhibited tumor growth, so the combination of starvation therapy, chemodynamic therapy and immunotherapy could achieve better therapeutic effect.

通过流式细胞术对肿瘤细胞上的PD-L1表达进行检测，实验结果表明，(4)组与(6)组肿瘤细胞上的PD-L1表达明显上调，所以实施例20得到的纳米酶可以引起肿瘤细胞上的PD-L1表达上调，增敏PD-L1抗体治疗，因此通过调控肿瘤代谢行为可以增强免疫治疗。The PD-L1 expression on tumor cells was detected by flow cytometry. The experimental results showed that the PD-L1 expression on tumor cells in groups (4) and (6) was significantly up-regulated, so the nanozyme obtained in Example 20 could It causes the up-regulation of PD-L1 expression on tumor cells and sensitizes PD-L1 antibody treatment, so immunotherapy can be enhanced by regulating tumor metabolic behavior.

实施例17-19、21-26得到的纳米酶与实施例20相似，可以抑制肿瘤生长，同时通过调控肿瘤代谢行为增强免疫治疗。Similar to Example 20, the nanozymes obtained in Examples 17-19 and 21-26 can inhibit tumor growth and enhance immunotherapy by regulating tumor metabolism.

以上实施例的只是用于帮助和理解本发明的方法其核心思想，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，可以对实施例进行改进和润饰，这些改进和润饰也应当视为本发明的保护范围。The above embodiments are only used to help and understand the core idea of the method of the present invention. For those skilled in the art, without departing from the principles of the present invention, the embodiments can be improved and modified. These improvements and retouching should also be regarded as the protection scope of the present invention.