基于类芬顿反应的Mn3O4/DOX@Lip纳米递药体系的构建及应用

doi:10.6023/A20120583

摘要/Abstract

利用肿瘤微环境与普通组织的差异, 我们设计了一种基于类芬顿反应(Fenton-like reaction)的微环境响应的纳米递药体系(Mn₃O₄/DOX@Lip). 首先利用热分解法制备得到锰的氧化物(Mn₃O₄)纳米粒子, 基于化疗药物阿霉素(DOX)与Mn原子的配位作用, 该纳米粒子可以负载并递送DOX, 其最外层包裹的酸敏脂质体能有效防止药物在递送过程中的泄露. 在肿瘤细胞的弱酸性和高浓度谷胱甘肽的微环境中, 脂质体膜破裂, 其中的Mn₃O₄纳米粒子被谷胱甘肽还原、分解, 并释放出DOX. 共聚焦成像结果表明, MCF-7细胞与Mn₃O₄/DOX@Lip共孵育后, 细胞中活性氧探针的荧光显著增加, 表明该递药体系可以通过下调细胞中的谷胱甘肽的含量引发细胞中的活性氧含量增加. 噻唑蓝(MTT)和流式细胞实验结果表明, 相比于单一的化疗药物, Mn₃O₄/DOX@Lip的细胞毒性增强, 这可能归因于还原产物Mn²⁺触发了类芬顿反应从而将细胞中内源性的H₂O₂转化为毒性更强的•OH. 氧化活性物质介导的细胞凋亡与化疗药物的细胞毒性相互促进, 增强了对细胞的凋亡能力. 本体系利用外源性的物质激发内源性的细胞毒性, 增强了化疗药物的治疗效果, 为肿瘤治疗提供了新的研究策略.

关键词: 锰的氧化物, 纳米递药体系, 类芬顿反应, 肿瘤微环境

The tumor microenvironment with low pH and high-level glutathione is different from other issues, which provides more possible strategies for designing drug delivery systems (DDSs). In this manuscript, we designed a microenvironment-responsive Mn₃O₄/DOX@Lip nano-DDS. Mn₃O₄ nanoparticles were prepared by thermal decomposition. Based on coordination of the chemotherapy drug doxorubicin (DOX) with Mn atom, the Mn₃O₄ nanoparticles could load and deliver DOX. A layer of sensitive liposomes was assembled on the outermost layer to further effectively prevent the leakage of DOX during the delivery process. X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDX) analysis results showed the manganese oxide was Mn₃O₄. The morphology and particle size of Mn₃O₄ were characterized by transmission electron microscope (TEM) and dynamic light scattering (DLS). Results showed that the Mn₃O₄ nanoparticles were a relatively uniform polyhedron with a particle size of around 20 nm. The drug loading of particles was 100 μg/mg. In weakly acidic microenvironment of tumor cells, the liposome membrane ruptured. In addition, the high-level glutathione microenvironment was also conducive to the decomposition of Mn ₃O₄ and the DOX was released in cells. Confocal imaging results demonstrated that after co-incubation with the Mn₃O₄ particles, the fluorescence of reactive oxygen species (ROS) probe in cancer cells increased significantly, indicating that the DDS could trigger an increase of ROS in cells by down-regulating the content of glutathione. The amount of ROS depended on the concentration of added particles and the incubation time. MTT and flow cytometry experiment results showed that Mn₃O₄/DOX@Lip had more cell cytotoxic compared to the single chemotherapy drug. It might be ascribed to the Fenton-like reaction triggered by the reduction product Mn²⁺, which converted the H₂O₂ in the cell into the more toxic •OH and promoted apoptosis. The ROS-mediated apoptosis and chemotherapy played a synergistic effect to enhance the ability of cell apoptosis. This proposed Mn ₃O₄/DOX@Lip nano-DDS uses exogenous substances to stimulate endogenous cytotoxicity and enhance the effect of chemotherapy drug, providing new research strategies for tumor treatment.

Key words: manganese oxide, nano-drug delivery system, Fenton-like reaction, tumor microenvironment

引用此文

蔡政, 张颖雯, 姜立萍, 朱俊杰. 基于类芬顿反应的Mn₃O₄/DOX@Lip纳米递药体系的构建及应用[J]. 化学学报, 2021, 79(4): 481-489.

Zheng Cai, Yingwen Zhang, Liping Jiang, Junjie Zhu. The Construction and Application of Mn₃O₄/DOX@Lip Nano-drug Delivery System Based on Fenton-Like Reaction[J]. Acta Chimica Sinica, 2021, 79(4): 481-489.

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图1. 肿瘤微环境响应的Mn3O4/DOX@Lip药物递送体系自组装过程和应用

Figure 1. The preparation and application of tumor microenvironment responsive Mn3O4/DOX@Lip drug delivery system

图2. Mn3O4纳米粒子的TEM图像: 在环己烷溶剂中(a); 用PAA处理后在水溶剂中(b); 包裹了脂质体后的Mn3O4@Lip (磷钨酸负染) (c); Mn3O4 纳米粒子的SEM图像(d); Mn3O4, Mn3O4/DOX和Mn3O4/DOX@Lip粒子在水溶液中的粒径分布图(e); Mn3O4, Mn3O4/DOX, 空白脂质体和Mn3O4/DOX@Lip的zeta电势(f)

Figure 2. TEM images of Mn3O4 nanoparticles: in cyclohexane solvent (a); after treated by PAA in water solvent (b); Mn3O4@Lip (phosphotungstic acid negative staining) (c); SEM image of Mn3O4 nanoparticles (d); hydrodynamic size distributions of Mn3O4, Mn3O4/DOX and Mn3O4/DOX@Lip particles in water (e); the zeta potential of Mn3O4, Mn3O4/DOX, blank Lip and Mn3O4/DOX@Lip (f)

图 3. Mn3O4粒子的XRD衍射图(a)和Mn3O4/OA和Mn3O4/PAA FT-IR谱图(b)

Figure 3. X-ray diffraction pattern of Mn3O4 particles (a) and FT-IR spectra of Mn3O4/OA and Mn3O4/PAA (b)

图4. Mn3O4/DOX中DOX的累积释放量, 在不同缓冲溶液(pH=7.4, 5.0)中(a); 在不同浓度的GSH溶液中(b); 在GSH溶液浓度均为10 μmol/mL, 不同缓冲溶液中: pH=5.0, pH=7.4 (c)

Figure 4. The cumulative release of DOX from Mn3O4/DOX (a) in different buffer solutions (pH=7.4, 5.0); in different concentration of GSH (b); in the same GSH solution (10 μmol/mL), with different buffer solution, pH=5.0, and pH=7.4 (c)

图5. Mn3O4纳米粒子在含GSH (10 μmol/mL)的缓冲溶液(pH=5.0)中2, 8和24 h的TEM图

Figure 5. TEM images of Mn3O4 nanoparticles in buffer solution (pH=5.0) including GSH (10 μmol/mL) for 2, 8 and 24 h

图6. Mn3O4用GSH处理后的上清液和H2O2反应, 在665 nm处亚甲基蓝溶液的吸光值随时间的变化

Figure 6. The absorption change of methylene blue solutions at 665 nm during treated with the supernatant and H2O2. The supernatant was got after Mn3O4 reacted with GSH

Figure 7. MTT法评估不同剂型的体外细胞毒性. MCF-7 细胞分别与Mn3O4和Mn3O4@Lip (a); Free-DOX, Mn3O4, Mn3O4/DOX和Mn3O4/DOX@Lip (b)共孵育48 h. *P<0.05,**P<0.01 (n=3) The cell cytotoxicity of MCF-7 treated with Mn3O4和Mn3O4@Lip (a); Free-DOX, Mn3O4, Mn3O4/DOX and Mn3O4/DOX@Lip (b) for 48 h, respectively. Error bars indicate s.d. (n=3). *P<0.05,**P<0.01 (two-tailed t-test)

图8. 流式分析含DOX 3 µg/mL, Mn 3O4 60 μg/mL的不同剂型与MCF-7细胞共孵育24 h诱导的细胞凋亡, 使用Annexin V-FITC/PI染色 (Ⅰ) 对照组; (Ⅱ) Free-DOX; (Ⅲ) Mn 3O4; (Ⅳ) Mn3O4/DOX; (Ⅴ) Mn3O4/DOX@Lip

Figure 8. Flow cytometry analysis of MCF-7 cells apoptosis induced by different formulations with DOX 3 µg/mL, Mn 3O460 μg/mL for 24 h, using Annexin V-FITC/PI staining, (Ⅰ) Control; (Ⅱ) Free-DOX; (Ⅲ) Mn 3O4; (Ⅳ) Mn3O4/DOX; (Ⅴ) Mn3O4/DOX@Lip

图9. MCF-7与Mn3O4/DOX@Lip共孵育12 h后的激光共聚焦成像图. 红色的荧光来自于复合物中的DOX, 蓝色的荧光来自于DAPI染色的细胞核, 确定复合物在细胞中的位置. 红色标尺为50 μm

Figure 9. Confocal microscopy images of MCF-7 cells incubated with Mn3O4/DOX@Lip for 12 h. Red ?uorescence was from DOX released from drug delivery system, and blue ?uorescence came from the cell nuclei stained by DAPI to local the site of the complex. The red scale bar is 50 μm

图10. MCF-7 细胞共聚焦成像图, (a)与20 μg/mL Mn 3O4分别共孵育2, 4, 8 h; (b)与不同浓度5, 10, 20 μg/mL Mn 3O4共孵育4 h. 绿色荧光来自于DCFH-DA探针. 红色标尺为50 μm, 绿色标尺为75 μm. 细胞内DCFH-DA荧光强度中值(MFI)(c)不同孵育时间; (d)不同Mn 3O4浓度. 使用Image J分析软件, 每次分析都不少于40个细胞

Figure 10. Confocal microscopy images of MCF-7 cells (a) incubated with 20 μg/mL Mn 3O4 for 2, 4, 8 h, respectively; (b) incubated with 5, 10, 20 μg/mL Mn 3O4 for 4 h, respectively. The green fluorescence came from the DCFH-DA probe. The red scale bar is 50 μm and the green scale bar is 75 μm. Quantitative median fluorescence intensity (MFI) of DCFH-DA in cells. Different incubation time (c); different concentration of Mn 3O4 (d). Use Image J to analyze at least 40 cells each time.

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基于类芬顿反应的Mn₃O₄/DOX@Lip纳米递药体系的构建及应用

The Construction and Application of Mn₃O₄/DOX@Lip Nano-drug Delivery System Based on Fenton-Like Reaction

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