通过针对纳米粒诱导细胞响应的蛋白质组学分析揭示纳米粒跨上皮细胞转运机制

doi:10.5246/jcps.2021.02.009

中国药学(英文版) ›› 2021, Vol. 30 ›› Issue (2): 107-118.DOI: 10.5246/jcps.2021.02.009

通过针对纳米粒诱导细胞响应的蛋白质组学分析揭示纳米粒跨上皮细胞转运机制

张箭¹^,², 秦蒙蒙¹^,², 杨丹³, 代文兵¹^,², 张华¹^,², 王学清¹^,², 何冰¹^,²^,^*(), 张强¹^,²^,^*()

1. 北京大学药学院天然药物及仿生药物国家重点实验室, 北京 100191
2. 北京大学药学院分子药剂学与新释药系统北京市重点实验室, 北京 100191
3. 陕西科技大学食品与生物工程学院, 陕西西安 710021

收稿日期:2020-07-11 修回日期:2020-08-10 接受日期:2020-08-19 出版日期:2021-02-28 发布日期:2021-02-27
通讯作者: 何冰, 张强
作者简介:
+ Tel.: +86-10-82802791, E-mail: zqdodo@bjmu.edu.cn
+ Tel.: +86-10-82805935, E-mail: hebingmumu@163.com
基金资助:
The National Key R&D Program of China (Grant No. 2017YFA0205600) and the National Natural Science Foundation of China (Grant No. 81690264, 81573359 and 81703441).

Proteomic analysis on cellular response induced by nanoparticles reveals the nano-trafficking pathway through epithelium

Jian Zhang¹^,², Mengmeng Qin¹^,², Dan Yang³, Wenbing Dai¹^,², Hua Zhang¹^,², Xueqing Wang¹^,², Bing He¹^,²^,^*(), Qiang Zhang¹^,²^,^*()

1 State Key Laboratory of Natural and Biomimetic Drugs,School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
2 Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
3 School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi'an 710021, China

Received:2020-07-11 Revised:2020-08-10 Accepted:2020-08-19 Online:2021-02-28 Published:2021-02-27
Contact: Bing He, Qiang Zhang

摘要/Abstract

摘要：

药物在口服药物递送的应用可以有效地促进药物在肠上皮细胞的吸收和转运。然而, 缺乏有效性和安全性的机制研究限制了它们在人体的最终转化。虽然囊泡转运已被证实为纳米药物转运的一般特征, 但其更深层次的分子机制尚不清楚。此外, 纳米药物的细胞转运是一个由不同细胞器和组分参与的动态过程。然而, 现有的研究大多只关注纳米药物的静态定位, 而忽视了纳米药物对细胞的动态生物学效应。在此, 我们制备了金纳米粒作为模型, 并培养上皮细胞单层, 在分子水平上探索纳米-生物相互作用。传统的药物抑制策略和亚细胞成像技术阐明了巨胞饮/内吞体/多囊泡体/溶酶体的转运路径。基于质谱的蛋白质组策略被用来识别和定量分析参与纳米药物胞内转运的蛋白质。研究表明, 与亚细胞结构、信号转导、能量转化和代谢调节相关的多种蛋白都受到纳米粒转运的调控。这些蛋白表达的改变阐明了细胞内蛋白的作用, 并验证了传统的发现。更重要的是, 揭示了细胞对纳米粒转运的反馈机制。我们相信, 这些新的调控机制将为纳米药物通过上皮屏障的有效转运提供新的策略。

关键词: 上皮细胞, 金纳米粒, 胞内转运, 蛋白组学, 细胞响应

Abstract:

The application of nanomedicines in oral drug delivery effectively promotes the drug absorption and transportation through enterocytes. Nevertheless, the absence of mechanism studies on efficacy and safety limits their final translation in humans. Although the vesicular trafficking has been verified as the general character for transport of nanomedicines, the deeper mechanism in molecular mechanism is still unclear. Moreover, the cellular transport of nanomedicines is a dynamic process involved by different organelles and components. However, most of existing studies just pay attention to the static location of nanomedicines, but neglect the dynamic biological effects on cells caused by them. Here, we prepared gold nanoparticles (AuNPs) as the model and cultured epithelial cell monolayer to explore the nano-bio interactions at the molecular level. The traditional pharmacological inhibition strategy and subcellular imaging technology elucidated the macropinocytosis/endosome/MVB/lysosome pathway during the transportation of AuNPs. Proteomics strategy based on mass spectrometry (MS) was utilized to identify and quantify proteins involved in the cellular transport of nanomedicines. Multiple proteins related to subcellular structure, signal transduction, energy transformation and metabolism regulation were demonstrated to be regulated by nanoparticle transport. These alterations of protein expression clarified the effects of intracellular proteins and verified the conventional findings. More importantly, it revealed a feedback mechanism of cells to the nano-trafficking. We believed that these new regulatory mechanisms provided new insights into the efficient transport of nanomedicines through epithelial barriers.

Key words: Epithelium, Gold nanoparticles, Intracellular transport, Proteomics, Cellular response

Supporting:

张箭, 秦蒙蒙, 杨丹, 代文兵, 张华, 王学清, 何冰, 张强. 通过针对纳米粒诱导细胞响应的蛋白质组学分析揭示纳米粒跨上皮细胞转运机制[J]. 中国药学(英文版), 2021, 30(2): 107-118.

Jian Zhang, Mengmeng Qin, Dan Yang, Wenbing Dai, Hua Zhang, Xueqing Wang, Bing He, Qiang Zhang. Proteomic analysis on cellular response induced by nanoparticles reveals the nano-trafficking pathway through epithelium[J]. Journal of Chinese Pharmaceutical Sciences, 2021, 30(2): 107-118.

图/表 11

Figure 1. The preparation process of pristine AuNPs and AuNPs (BSA coating).

Figure 2. The shape and surface morphology of pristine AuNPs (A) and AuNPs (BSA coating) (B) by TEM. Both (A) and (B) were stained with uranyl acetate. Scale bar: 200 nm.

Figure 3. Distribution of the hydrodynamic diameter (A) and zeta potential (B) of pristine AuNPs (red) and AuNPs (BSA coating) (blue) detected by DLS.

Figure 4. UV-vis absorbance spectra of pristine AuNPs (red) and AuNPs (BSA coating) (blue).

Figure 5. Different endocytosis inhibitors were co-incubated with AuNPs in Caco-2 cells to explore the uptake pathway. The amount of AuNPs-trans without inhibitor addition was normalized as 100% and set as control. n = 3. In this study, all the quantitative data were shown as mean ± SD, whereas P values were determined by the two-sided student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.005 and ****P < 0.001).

Figure 6. (A) TEM observation of AuNPs in Caco-2 cells. Scale bar: 2 μm. (B) TEM observation of AuNPs in endosome of Caco-2 cells with enlarged structural details. Scale bar: 500 nm. (C) TEM observation of AuNPs in MVB of Caco-2 cells with enlarged structural details. Scale bar: 500 nm. (D) TEM observation of AuNPs in lysosome of Caco-2 cells with enlarged structural details. Scale bar: 500 nm. (E) TEM observation of AuNPs in ER of mouse small intestine tissue with enlarged structural details. Scale bar: 1 μm. (F) TEM observation of AuNPs in Golgi apparatus of mouse small intestine tissue with enlarged structural details. Scale bar: 1 μm.

Figure 7. The correlation analysis of cellular proteins investigated by LFQ proteomics after AuNPs incubated with Caco-2 cells.

Figure 8. Hierarchical clustering analysis of cellular proteins investigated by LFQ proteomics after AuNPs incubated with Caco-2 cells.

Figure 9. The Volcano plot of cellular proteins investigated by LFQ proteomics after AuNPs incubated with Caco-2 cells.

Figure 10. The GO analysis of cellular up-regulated proteins investigated by LFQ proteomics after AuNPs incubated with Caco-2 cells.

Figure 11. The GO analysis of cellular down-regulated proteins investigated by LFQ proteomics after AuNPs incubated with Caco-2 cells.

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通过针对纳米粒诱导细胞响应的蛋白质组学分析揭示纳米粒跨上皮细胞转运机制

Proteomic analysis on cellular response induced by nanoparticles reveals the nano-trafficking pathway through epithelium

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