WO2023070885A1 - 一种铁死亡诱导剂及其制备方法和应用 - Google Patents

一种铁死亡诱导剂及其制备方法和应用 Download PDF

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WO2023070885A1
WO2023070885A1 PCT/CN2021/138481 CN2021138481W WO2023070885A1 WO 2023070885 A1 WO2023070885 A1 WO 2023070885A1 CN 2021138481 W CN2021138481 W CN 2021138481W WO 2023070885 A1 WO2023070885 A1 WO 2023070885A1
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vermiculite
nanosheets
ferroptosis
ferroptosis inducer
preparation
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PCT/CN2021/138481
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French (fr)
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张鹏飞
于兴华
蔡林涛
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A61K33/12—Magnesium silicate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention belongs to the technical field of preparation of antitumor drugs, and in particular relates to a ferroptosis inducer and its preparation method and application.
  • cancer With increasing morbidity and mortality, cancer has become a major global public health problem.
  • the main treatment methods for cancer include surgery, chemotherapy, radiotherapy, immunotherapy, etc., but these commonly used treatment methods also have some disadvantages, such as surgery is prone to recurrence, radiotherapy is not ideal due to radiation resistance, and chemotherapy is due to drug selection. Poor sex can have serious side effects. Therefore, emerging cancer treatment methods such as biological therapy are also constantly emerging in order to solve the problems existing in the prior art.
  • Ferroptosis is a new type of programmed cell death caused by the accumulation of iron-dependent lipid peroxides, which was first proposed by Professor Brent R. Stockwell in 2012. Recent studies have shown that induction of ferroptosis can be used in cancer therapy, especially in the eradication of aggressive malignancies that are resistant to conventional therapies. With the development of nanobiotechnology, ferroptosis-based anticancer nanomedicine has also made important progress, which are mainly divided into two categories: iron-based nanomaterials and non-iron-based nanomaterials.
  • Iron-based nanomaterials can enrich iron ions in cells and accelerate the Fenton reaction, thereby increasing the level of reactive oxygen species (ROS) in cells and inducing ferroptosis.
  • the cisplatin-loaded iron oxide nanoprodrug (FePt NP2) constructed by Ma et al. can release cisplatin and Fe 2+ /Fe 3+ at a specific tumor site, and the Fenton reaction occurs in situ, which significantly increases the intracellular ROS level, thereby Induces tumor cell ferroptosis and enhances anticancer activity.
  • the iron-chelating semiconductor polycomposite nanoparticles (SPFeN) reported by Pu et al. combine photothermal therapy with ferroptosis therapy to enhance the therapeutic effect of cancer.
  • iron-based nanomaterials such as iron oxide nano-prodrugs loaded with cisplatin and iron-chelated semiconductor multi-composite nanoparticles enhance the anti-tumor effect by inducing ferroptosis, they often need to use higher iron doses or combine them with other treatments. Therefore, it has a complex nanostructure and multi-metal components, and its biological safety is not high.
  • Non-iron-based nanomaterials can inhibit glutathione peroxidase (GPX4) or induce ferroptosis by increasing the degree of lipid peroxidation in tumor cells through exogenous regulation.
  • Gao et al. used amphiphilic polymer micelles to deliver the GPX4 inhibitor RSL3, and reversed multidrug resistance by synergistically inducing ferroptosis through GPX4 inhibition, glutathione (GSH) attenuation, and lipid peroxidation.
  • GSH glutathione
  • Arginine-rich manganese-silicon nanobubbles prepared by Wang et al. can be used as a ferroptosis inducer to achieve tumor-targeted therapy through intracellular GSH depletion.
  • polymer micelles loaded with small molecule ferroptosis inducers and amorphous calcium carbonate composite nano-drugs disclosed in the prior art can effectively reverse drug resistance or further kill tumor cells by inducing ferroptosis, there is drug leakage. with the risk of toxic side effects.
  • manganese-silicon nanobubbles with GSH depletion ability can realize tumor diagnosis and treatment at the same time, they can only target arginine succinate synthase-deficient tumor cells, and are not universal to tumor cells.
  • the residual nanomaterials in the body may have the risk of long-term toxicity and so on.
  • the present invention proposes a ferroptosis inducer and its preparation method and application.
  • the ferroptosis inducer provided by the present invention is absorbed by hypoxic tumor cells, the redox couple (Fe 2+ /Fe 3+ ) can generate OH and O 2 through the disproportionation reaction of hydrogen peroxide and the Fenton reaction, and can supply oxygen independently .
  • the ferroptosis inducer of the present invention can regulate the tumor microenvironment (TME) by depleting glutathione, which can induce ferroptosis in tumor cells, and thus is suitable for the preparation of ferroptosis-based anticancer nano-medicines.
  • TAE tumor microenvironment
  • a ferroptosis inducer comprising vermiculite nanoplatelets.
  • the thickness of the vermiculite nanosheets is 1.0-1.3 nm.
  • the length of the vermiculite nanosheets is 305-335nm.
  • the width of the vermiculite nanosheets is 305-335nm.
  • the present invention also provides a preparation method for the above-mentioned ferroptosis inducer, which includes obtaining vermiculite nanosheets; the vermiculite nanosheets are obtained by adding vermiculite to an alkali metal ion salt solution for intercalation and exfoliation; wherein, each mg of Vermiculite is added with 0.1-10 moles per liter of alkali metal salt modifier.
  • each mg of Vermiculite is added with 0.1-10 moles per liter of alkali metal salt modifier.
  • the concentration of the added alkali metal salt modifier is lower than 0.1 mole per liter, the thickness of the prepared vermiculite sheet will be thickened; if the concentration of the alkali metal modifier is higher than 10 moles per liter, longer wash times are required to remove the alkali metal modifier.
  • the concrete preparation method of described vermiculite nanosheet comprises the following steps:
  • the alkali metal salt solution modifier is one or more of lithium salt solution, potassium salt solution or sodium salt solution.
  • the alkali metal salt solution modifier is one or more of lithium chloride solution, lithium ethylenediaminetetraacetate solution or lithium citrate solution.
  • the chemically expanded vermiculite treated with the lithium salt modifier compared with the thermally expanded or hydrogen peroxide-expanded physically expanded vermiculite, the vermiculite flakes can be exfoliated more completely and thinner.
  • Lithium ethylenediamine tetraacetate and lithium citrate modifiers have the best expansion effect on vermiculite, which can reduce the order degree of vermiculite-phlogopite mixed layer minerals and phlogopite crystals in vermiculite, and vermiculite can be stripped more completely.
  • step S1 is heating under reflux at a temperature of 80-90° C. for 24-36 hours.
  • step S3 the duration of ultrasonic treatment is 0.3-0.6h.
  • the present invention also provides the application of the above-mentioned ferroptosis inducer as an antitumor drug in regulating tumor microenvironment.
  • the ferroptosis inducer provided by the invention has good biocompatibility and strong cancer cell killing effect, can be used in the synthesis of tumor treatment drugs, and has good application prospects.
  • FIG. 1 is the absorption spectrum of TMB under different H2O2 concentrations
  • Figure 2 shows the change of absorbance at 650 nm under different H 2 O 2 concentrations
  • Figure 3 is the absorption spectrum of DTNB under different GSH concentrations
  • Figure 4 shows the change of absorbance at 412 nm under different GSH concentrations
  • Fig. 5 is the iron content measurement result in the MC38 cell of embodiment 4.
  • Fig. 6 is the detection result of GPX4 activity in the MC38 cell of embodiment 5;
  • FIG. 7 is the result of tumor tissue analysis in Example 6.
  • a ferroptosis inducer comprising vermiculite nanoplatelets.
  • the vermiculite nano sheet is obtained by adding vermiculite into alkali metal ion salt solution for intercalation treatment and stripping, wherein 0.1-10 moles per liter of alkali metal salt modifier is added to every milligram of vermiculite.
  • the vermiculite nanosheets have a thickness of 1.0 nm, a length of 310 nm, and a width of 315 nm.
  • Concrete preparation method comprises the following steps:
  • the ferroptosis inducer provided in this example can be used as an antitumor drug to regulate the tumor microenvironment to achieve antitumor effect.
  • a ferroptosis inducer comprising vermiculite nanoplatelets.
  • the vermiculite nano sheet is obtained by adding vermiculite to alkali metal ion salt solution for intercalation treatment, wherein 0.1-10 moles per liter of alkali metal salt modifier is added to every milligram of vermiculite.
  • the vermiculite nanosheets have a thickness of 1.2 nm, a length of 305 nm, and a width of 335 nm.
  • Concrete preparation method comprises the following steps:
  • the ferroptosis inducer provided in this example can be used as an antitumor drug to regulate the tumor microenvironment to achieve antitumor effect.
  • a ferroptosis inducer including vermiculite nanosheets, vermiculite nanosheets are obtained by adding vermiculite to alkali metal ion salt solution for intercalation treatment, and exfoliating, wherein XX moles of alkali metal salt are added to every mg of vermiculite Modifier.
  • the vermiculite nanosheets have a thickness of 1.3nm, a length of 335nm, and a width of 305nm.
  • Concrete preparation method comprises the following steps:
  • the ferroptosis inducer provided in this example can be used as an antitumor drug to regulate the tumor microenvironment to achieve antitumor effects.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • the detection principle is: vermiculite nanosheets act as a peroxidase, catalyzing TMB to produce a soluble blue product, and at the same time catalyzing H 2 O 2 into H 2 O, the blue product of TMB can usually measure the absorbance at 620-650nm.
  • TMB solution 100 ⁇ g/mL was mixed with H2O2 (0, 1, 2.5, 5, 10 and 30 mM).
  • H2O2 0., 1, 2.5, 5, 10 and 30 mM
  • NSs 200 ⁇ g/mL
  • absorbance peaks are collected and the H2O2 concentration is plotted.
  • the numbers 1-6 represent the absorbance results of the test groups with H 2 O 2 concentrations of 30mM, 10mM, 5mM, 2.5mM, 1mM, and 0mM, respectively.
  • the OH produced by the degradation of hydrogen peroxide catalyzed by vermiculite nanosheets can oxidize TMB, showing blue oxTMB, calculated by plotting the substrate concentration and reaction rate of absorbance at 650 nm and fitting to Michaelis-Menton kinetic analysis, Km and
  • the maximum reaction velocity (Vmax) is 3.4mM and 7.97 ⁇ 10 -8 Ms -1 , which proves that the enzyme activity of the vermiculite nanosheets is comparable to that of peroxidase mimetic enzymes in the prior art.
  • the detection mechanism is: vermiculite nanosheets act as glutathione oxidase, and when glutathione GSH is oxidized, DTNB will appear yellow.
  • the numbers 1-6 represent the absorbance results of the test groups with GSH concentrations of 0mM, 0.0625mM, 0.125mM, 0.25mM, 0.5mM, and 1mM, respectively.
  • Vmax maximum reaction velocity
  • Iron content in MC38 cells incubated with different treatments was detected by iron colorimetric kit (Applygen, E1042).
  • NSs medium 100 ⁇ g/mL was added to the 6-well plate, and the cells were incubated for 12 h. Finally, detect the iron content according to the method used in the kit.
  • Cell peroxidase detection kit (Beyotime, S0056) was used to detect GPX4 activity in MC38 cells incubated with different treatments. Vermiculite nanosheet medium (100 ⁇ g/mL) was added to the 6-well plate, and then incubated for 0-12 h. Finally, detect GPX4 activity according to the method used in the kit.
  • GPX4 glutathione peroxidase 4
  • GPX4 glutathione peroxidase 4
  • NSs is a glutathione oxidase-mimicking enzyme that leads to glutathione depletion.
  • NSs can also cause intracellular iron overload and induce ferroptosis.
  • Cellular GPX4 activity was detected using a cellular glutathione peroxidase assay kit. Compared with the control group (control group) and the DCPy group, GPX4 activity was significantly decreased in the NSs-associated group.

Abstract

一种铁死亡诱导剂及其制备方法和应用。铁死亡诱导剂包括蛭石纳米片，所述蛭石纳米片的厚度为1.0-1.3nm，长度为305-335nm，宽度为305-335nm。所述的铁死亡诱导剂具有生物相容性好以及很强的癌细胞杀伤效果，可用于肿瘤治疗药物的合成，具有很好的应用前景。

Description

一种铁死亡诱导剂及其制备方法和应用 技术领域
本发明属于抗肿瘤药物制备技术领域，具体涉及一种铁死亡诱导剂及其制备方法和应用。
 背景技术
随着发病率和死亡率的不断增加，癌症已经成为一个重大的全球公共卫生问题。目前癌症主要的治疗方式有手术，化疗，放疗，免疫治疗等，但是这些常用的治疗方式也存在着一些弊端，比如手术易复发，放疗因辐射抵抗的问题使得放疗效果不理想，化疗因药物选择性差会带来严重的副作用。因此，生物治疗等新兴的癌症治疗方式为了解决现有技术中存在的问题也在不断涌现。
铁死亡是一种由铁依赖的脂质过氧化物的积累导致细胞死亡的新型程序性细胞死亡方式，由Brent R. Stockwell教授于2012年首次提出。近期研究表明，诱导铁死亡可以用于癌症治疗，尤其在根除对传统疗法有耐药性的侵袭性恶性肿瘤。随着纳米生物技术的发展，基于铁死亡的抗癌纳米药物也取得了重要进展，主要分为铁基纳米材料和非铁基纳米材料两大类。
铁基纳米材料能够在细胞内富集铁离子，加快芬顿(Fenton)反应，从而提高细胞内活性氧(ROS)水平，诱导铁死亡。如Ma等构建的负载顺铂的氧化铁纳米前药(FePt NP2)可以在肿瘤特定部位释放出顺铂和Fe 2+/Fe 3+，原位发生Fenton反应，显著提高细胞内ROS水平，从而诱导肿瘤细胞铁死亡，增强抗癌活性。Pu等报道的铁螯合半导体多复合纳米颗(SPFeN)将光热疗法与铁死亡疗法相结合，加强了癌症治疗效果。但是，负载顺铂的氧化铁纳米前药和铁螯合 半导体多复合纳米颗粒等铁基纳米材料虽然通过诱导铁死亡效应增强了抗肿瘤效果，但是往往需要使用较高的铁剂量或者与其他治疗方式联合使用，因此具有复杂的纳米结构和多金属成分，生物安全性不高。
非铁基纳米材料可以抑制谷胱甘肽过氧化物酶(GPX4)或者通过外源性调节增加肿瘤细胞脂质过氧化程度来诱导铁死亡。Gao等利用两亲性聚合物胶束递送GPX4抑制剂 RSL3，通过GPX4抑制、谷胱甘肽(GSH)衰减及脂质过氧化三者协同诱导铁死亡逆转多药耐药。Wang等制备的富精氨酸的锰硅纳米泡可以通过细胞内GSH耗竭作为铁死亡诱导剂实现肿瘤靶向诊疗。然而，包载小分子铁死亡诱导剂的聚合物胶束以及现有技术公开的无定型碳酸钙复合纳米药物虽然可以通过诱导铁死亡有效逆转耐药或者联合化疗进一步杀伤肿瘤细胞，但是存在药物泄露与毒副作用的风险。具有GSH消耗能力的锰硅纳米泡虽然可以同时实现肿瘤的诊断与治疗，但只能针对精氨酸琥珀酸合成酶缺陷的肿瘤细胞，对肿瘤细胞不具有普适性。此外，体内残留的纳米材料可能存在长期毒性的风险等。
因此，研究开发一种对肿瘤细胞具有普适性且无细胞毒性风险的超薄蛭石纳米片来诱导铁死亡，将具有良好的应用前景和广阔的适用性。
 技术问题
为了克服现有技术存在的缺陷，本发明提出了一种铁死亡诱导剂及其制备方法和应用。本发明提供的铁死亡诱导剂一旦被缺氧的肿瘤细胞吸收，氧化还原对(Fe 2+/Fe 3+)通过过氧化氢的歧化反应以及Fenton反应生成·OH和O 2，能够自主补充氧气。此外，本发明的铁死亡诱导剂能够通过消耗谷胱甘肽来调节肿瘤微环境（TME），谷胱甘肽可诱导肿瘤细胞的铁下垂，因此适用于制备基于铁死亡的抗癌纳米药物。
技术解决方案
一种铁死亡诱导剂，包括蛭石纳米片。
进一步地，所述蛭石纳米片的厚度为1.0-1.3nm。
进一步地，所述蛭石纳米片的长度为305-335nm。
进一步地，所述蛭石纳米片的宽度为305-335nm。
本发明还提供上述铁死亡诱导剂的制备方法，包括获取蛭石纳米片；所述蛭石纳米片由蛭石加入到碱金属离子盐溶液进行插层处理，经剥离得到；其中，每毫克的蛭石加入0.1-10摩尔每升的碱金属盐改性剂。每毫克的蛭石中，加入的碱金属盐改性剂的浓度若低于0.1摩尔每升，则会导致制得的蛭石片厚度变厚；若碱金属改性剂的浓度高于10摩尔每升，则需要更长的洗涤时间将碱金属改性剂洗去。
所述蛭石纳米片的具体制备方法包括以下步骤：
S1：将蛭石加入到碱金属盐溶液改性剂中，对蛭石进行膨胀；
S2：膨胀后进行分离，收集上层胶状浆液；
S3：将上层胶状浆液进行超声处理，剥离得到蛭石纳米片。
优选地，所述碱金属盐溶液改性剂为锂盐溶液、钾盐溶液或钠盐溶液中的一种或多种。
更优选地，所述碱金属盐溶液改性剂为氯化锂溶液、乙二胺四乙酸锂溶液或柠檬酸锂溶液中的一种或多种。采用锂盐改性剂处理得到的化学法膨胀蛭石，与热膨胀或双氧水膨胀的物理法蛭石相比，蛭石片可以剥离地更完全，厚度更薄。乙二胺四乙酸锂,柠檬酸锂改性剂对蛭石的膨胀作用效果最好，可以使蛭石中的蛭石-金云母混层矿物和金云母等晶体的有序度降低，蛭石能够被剥离得更完全。
进一步地，步骤S1的条件为在温度为80-90℃下回流加热24-36h。
进一步地，步骤S3中，超声处理的时长为0.3-0.6h。
本发明还提供上述铁死亡诱导剂作为抗肿瘤药物在调节肿瘤微环境中的应用。
 有益效果
本发明的有益效果包括：
本发明提供的铁死亡诱导剂具有生物相容性好以及很强的癌细胞杀伤效果，可用于肿瘤治疗药物的合成，具有很好的应用前景。
 附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为不同H 2O 2浓度下TMB的吸收光谱图；
图2为不同H 2O 2浓度下650 nm处的吸光度变化情况；
图3为不同GSH浓度下DTNB的吸收光谱图；
图4为不同GSH浓度下412 nm处的吸光度变化情况；
图5为实施例4的MC38细胞内铁含量测定结果；
图6为实施例5的MC38细胞内GPX4活性检测结果；
图7为实施例6的肿瘤组织分析结果。
 本发明的实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的一部分实施例，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。
实施例1
一种铁死亡诱导剂，包括蛭石纳米片。蛭石纳米片由蛭石加入到碱金属离子盐溶液进行插层处理，经剥离得到，其中，每毫克的蛭石加入0.1-10摩尔每升的碱金属盐改性剂。蛭石纳米片的厚度为1.0nm，长度为310nm，宽度为315nm。
具体制备方法包括如下步骤：
S1：将50mg商用蛭石粉加入到5摩尔每升的氯化锂溶液中，在80℃下回流加热24 h，对蛭石粉进行插层处理，使之膨胀；
S2：膨胀结束后，以3000rpm的转速离心20分钟，收集上层胶状浆液；
S3：对上层胶状浆液进行多次超声处理，超声处理的时长为0.5h，剥离得到蛭石纳米片。
本实施例提供的铁死亡诱导剂可作为抗肿瘤药物用于调节肿瘤微环境，实现抗肿瘤的效果。
实施例2
一种铁死亡诱导剂，包括蛭石纳米片。蛭石纳米片由蛭石加入到碱金属离子盐溶液进行插层处理得到，其中，每毫克的蛭石加入0.1-10摩尔每升的碱金属盐改性剂。蛭石纳米片的厚度为1.2nm，长度为305nm，宽度为335nm。
具体制备方法包括如下步骤：
S1：将50mg商用蛭石粉加入到100摩尔每升的乙二胺四乙酸锂溶液中，在85℃下回流加热24 h，对蛭石粉进行插层处理，使之膨胀；
S2：膨胀结束后，以3500rpm的转速离心30分钟，收集上层胶状浆液；
S3：对上层胶状浆液进行多次超声处理，超声处理的时长为0.3h，剥离得到蛭石纳米片。
本实施例提供的铁死亡诱导剂可作为抗肿瘤药物用于调节肿瘤微环境，实现抗肿瘤的效果。
实施例3
一种铁死亡诱导剂，包括蛭石纳米片，蛭石纳米片由蛭石加入到碱金属离子盐溶液进行插层处理，经剥离得到，其中，每毫克的蛭石加入XX摩尔的碱金属盐改性剂。蛭石纳米片的厚度为1.3nm，长度为335nm，宽度为305nm。
具体制备方法包括如下步骤：
S1：将50mg商用蛭石粉加入到500摩尔每升的柠檬酸锂溶液中，在90℃下回流加热32 h，对蛭石粉进行插层处理，使之膨胀；
S2：膨胀结束后，以3000rpm的转速离心25分钟，收集上层胶状浆液；
S3：对上层胶状浆液进行多次超声处理，超声处理的时长为0.6h，剥离得到蛭石纳米片。
本实施例提供的铁死亡诱导剂可作为抗肿瘤药物用于调节肿瘤微环境，实现抗肿瘤的效果。
实验例1  蛭石纳米片的过氧化物酶模拟活性评价
在温度为37℃、 pH 为6.5的PBS溶液中，以3,3'，5,5' -四甲基联苯胺（TMB）为染色指示剂进行蛭石纳米片（NSs）降解H 2O 2的过氧化物酶模拟活性评价。
检测原理为：蛭石纳米片起过氧化物酶的作用，催化TMB产生可溶性蓝色产物，同时将H 2O 2催化成H 2O，TMB的蓝色产物通常可以在620-650nm测定吸光度。
Figure dest_path_image001
TMB溶液(100 μg/mL)与H 2O 2(0、1、2.5、5、10和30 mM)混合。加入NSs(200 μg/mL)，在37℃下反应15分钟，进行稳态动力学分析。为此，采集吸光度峰值，并绘制H 2O 2浓度图。此外，利用ε= 39000 M -1cm -1对oxTMB进行线性Lineweaver-Burk绘图，以确定Km和Vmax。
结果参见图1和图2，图1中，标号1-6分别代表H 2O 2浓度为30mM、10mM、5mM、2.5mM、1mM、0mM的测试组的吸光度结果。蛭石纳米片催化过氧化氢降解产生的·OH可以氧化TMB，呈现蓝色oxTMB，通过绘制吸光度的底物浓度与反应速率计算值在650 nm和拟合到Michaelis-Menton动力学分析, Km和最大反应速度(Vmax)为3.4mM和7.97×10 -8Ms -1，证明蛭石纳米片的酶活力与现有技术中的过氧化物酶模拟酶具有可比性。
实验例2  蛭石纳米片的谷胱甘肽氧化酶模拟活性评价
蛭石纳米片谷胱甘肽氧化酶模拟活性评价以5'-二硫代二(2-硝基苯甲酸)（DTNB）为指示剂，以谷胱甘肽（GSH）为底物进行蛭石纳米片的谷胱甘肽氧化酶模拟活性评价。
检测机理为：蛭石纳米片起谷胱甘肽氧化酶的作用，氧化谷胱甘肽GSH时，DTNB将显黄色。
Figure dest_path_image002
DTNB在DMSO (300 μg/mL)中与GSH(0, 0.0625, 0.125, 0.25, 0.5和1 mM)混合。加入NSs(200 μg/mL)，在37℃下反应5分钟，进行稳态动力学分析。采集0-15分钟的吸光度并绘制谷胱甘肽浓度图。此外，利用TNB的ε= 13600 M -1cm -1进行线性Lineweaver-Burk绘图，以确定Km和Vmax。
结果见图3、4，图3中，标号1-6分别代表GSH浓度为0mM、0.0625mM、0.125mM、0.25mM、0.5mM、1mM的测试组的吸光度结果。通过绘制吸光度的底物浓度与反应速率计算值在412 nm和拟合到Michaelis-Menton动力学分析, Km和最大反应速度(Vmax)为1.34mM和1.35×10 -4Ms -1，证明蛭石纳米片的酶活力与现有技术中的谷胱甘肽氧化模拟酶具有可比性
实验例3  细胞内铁含量测定
采用铁比色试剂盒(Applygen, E1042)检测不同处理孵育的MC38细胞内铁含量。在6孔板中加入NSs培养基(100 μg/mL)，然后细胞孵育12 h。最后，按照试剂盒使用方法检测铁含量。
结果见图5，与PBS(control组)或DCPy组相比，NSs相关组检测到更多的铁离子，表明NSs诱导细胞内铁超载，诱发铁死亡。
实验例4  细胞内GPX4活性测定
使用细胞过氧化物酶检测试剂盒(Beyotime, S0056)检测不同处理孵育的MC38细胞内GPX4活性。将蛭石纳米片培养基(100 μg/mL)加入到的6孔板中，然后孵育0-12 h。最后，按照试剂盒使用方法检测GPX4活性。
结果见图6，谷胱甘肽过氧化物酶4 (GPX4)作为铁下垂的中心调控因子，在脂质修复系统中具有重要作用，谷胱甘肽消耗可使GPX4失活，从而诱导铁死亡。NSs是一种谷胱甘肽氧化酶模拟酶，导致谷胱甘肽耗竭。此外，NSs还可引起细胞内铁超载，诱发铁死亡。使用细胞谷胱甘肽过氧化物酶检测试剂盒检测细胞GPX4活性。与对照组（control组）和DCPy组相比，NSs相关组GPX4活性显著降低。这些结果表明，NSs通过抑制GPX4活性显著诱导了铁死亡。
实验例5  肿瘤组织分析
设置4组实验，分别是PBS组、PBS+light（光照射）组、NSs组和NSs+light（光照射）组，GPX4抗体对不同处理的肿瘤切片进行染色。随后用DAPI对细胞核进行染色。这些肿瘤切片由Olympus显微镜(SLIDEVIEW VS200)拍摄。
结果见图7，肿瘤组织切片免疫组化染色结果显示NSs组GPX4明显下调（NSs组的褐色部分比PBS组明显减少），说明治疗效果显著归因于铁死亡。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种铁死亡诱导剂，其特征在于，包括蛭石纳米片。
  2. 根据权利要求1所述的铁死亡诱导剂，其特征在于，所述蛭石纳米片的厚度为1.0-1.3nm。
  3. 根据权利要求1所述的铁死亡诱导剂，其特征在于，所述蛭石纳米片的长度为305-335nm。
  4. 根据权利要求1所述的铁死亡诱导剂，其特征在于，所述蛭石纳米片的宽度为305-335nm。
  5. 一种根据权利要求1-4任一项所述的铁死亡诱导剂的制备方法，其特征在于，包括获取蛭石纳米片；所述蛭石纳米片由蛭石加入到碱金属离子盐溶液进行插层处理，经剥离得到；其中，每毫克的蛭石加入0.1-10摩尔每升的碱金属盐改性剂。
  6. 根据权利要求5所述的铁死亡诱导剂的制备方法，其特征在于，
    所述蛭石纳米片的具体制备方法包括以下步骤：
    S1：将蛭石加入到碱金属盐溶液改性剂中，对蛭石进行膨胀；
    S2：膨胀后进行分离，收集上层胶状浆液；
    S3：将上层胶状浆液进行超声处理，剥离得到蛭石纳米片。
  7. 根据权利要求5或6所述的铁死亡诱导剂的制备方法，其特征在于，所述碱金属盐溶液改性剂为锂盐溶液、钾盐溶液或钠盐溶液中的一种或多种。
  8. 根据权利要求5或6所述的铁死亡诱导剂的制备方法，其特征在于，所述碱金属盐溶液改性剂为氯化锂溶液、乙二胺四乙酸锂溶液或柠檬酸锂溶液中的一种或多种。
  9. 根据权利要求6所述的铁死亡诱导剂的制备方法，其特征在于，步骤S3中，超声处理的时长为0.3-0.6h。
  10. 根据权利要求1-4任一项所述的铁死亡诱导剂或根据权利要求4或5-9任一项方法制备得到的铁死亡诱导剂作为抗肿瘤药物在调节肿瘤微环境中的应用。
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