基于DNA纳米花的细胞自噬基因沉默用于增敏抗肿瘤化疗

doi:10.7503/cjcu20200242

高等学校化学学报 ›› 2020, Vol. 41 ›› Issue (7): 1461.doi: 10.7503/cjcu20200242

• 庆祝《高等学校化学学报》复刊40周年专栏 • 上一篇下一篇

基于DNA纳米花的细胞自噬基因沉默用于增敏抗肿瘤化疗

张开翔^1,²,刘军杰¹,宋巧丽¹,王丹钰¹,史进进^1,^*(),张海悦^3,^*(),李景虹^2,^*()

1.郑州大学药学院, 河南省肿瘤重大疾病靶向治疗与诊断重点实验室, 郑州450001
2.清华大学化学系, 北京100084
3.长春工业大学化学与生命科学学院, 长春130012

收稿日期:2020-04-29 出版日期:2020-07-10 发布日期:2020-06-02
通讯作者: 史进进,张海悦,李景虹 E-mail:shijinyxy@zzu.edu.cn;zhhaiyue@163.com;jhli@mail.tsinghua.edu.cn
基金资助:
国家自然科学基金(21904119);国家自然科学基金(21621003);河南省科技攻关项目(192102310147)

Multifunctional DNA Nanoflowers for Autophagy Inhibition and Enhanced Antitumor Chemotherapy^†

ZHANG Kaixiang^1,²,LIU Junjie¹,SONG Qiaoli¹,WANG Danyu¹,SHI Jinjin^1,^*(),ZHANG Haiyue^3,^*(),LI Jinghong^2,^*()

1. Key laboratory of Targeting Therapy and Diagnosis for Critical Diseases, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
2. Department of Chemistry, Tsinghua University, Beijing 100084, China
3. College of Chemical and Life Science, Changchun University of Technology, Changchun 130012, China

Received:2020-04-29 Online:2020-07-10 Published:2020-06-02
Contact: Jinjin SHI,Haiyue ZHANG,Jinghong LI E-mail:shijinyxy@zzu.edu.cn;zhhaiyue@163.com;jhli@mail.tsinghua.edu.cn
Supported by:
† National Natural Science Foundation of China(21904119);National Natural Science Foundation of China(21621003);Key Scientific Research Projects of Science and Technology Department of Henan Province, China(192102310147)

摘要/Abstract

摘要：

自噬是真核细胞降解蛋白质的重要途径之一, 在细胞的更新代谢中起重要作用. 肿瘤细胞借助高水平的细胞自噬能够阻断细胞凋亡途径, 降低化疗药物的抗肿瘤效果. 本文通过设计编码有核酸适配体序列(Aptamer)和DNA酶序列(DNAzyme)的多功能DNA纳米花, 利用DNA序列可负载化疗药物阿霉素(Dox)的特性, 实现了对肿瘤细胞特异靶向的药物递送, 并高效沉默肿瘤细胞的自噬相关基因ATG5, 达到增敏抗肿瘤化疗的效果. 通过RT-PCR实验验证合成的DNA纳米花可以有效剪切肿瘤细胞中自噬相关基因ATG5的mRNA; 并通过DNA纳米花的细胞毒性和细胞凋亡实验研究了其对肿瘤细胞系MCF-7的靶向治疗作用, 结果显示该多功能DNA纳米花在增敏抗肿瘤化疗方面具有明显优势.

关键词: DNA纳米花, 药物递送, 自噬, DNA酶, 抗肿瘤化疗

Abstract:

Autophagy is an important process in eukaryotic cells, in which subcellular membranes undergo dynamic morphological changes, leading to the degradation of cellular proteins and cytoplasmic organelles. High levels of cellular autophagy could protect tumor cells from apoptosis and reduce the antitumor effect of chemotherapy drugs. In this work, we designed a multifunctional DNA nanoflower, which encode DNA aptamer and DNAzyme for targeted drug delivery and gene silencing to inhibit autophagy in tumor cells. Using RT-PCR, we verified that the designed DNA nanoflower can effectively cleave the mRNA of autophagy-related gene(ATG5) in tumor cells. The cytotoxicity and cell apoptosis assay also showed that the synthesized multifunctional DNA nanoflower could enhance antitumor chemotherapy.

Key words: DNA nanoflowers, Drug delivery, Autophagy, DNAzyme, Antitumor chemotherapy

中图分类号:

O651
O636.9

TrendMD:

张开翔, 刘军杰, 宋巧丽, 王丹钰, 史进进, 张海悦, 李景虹. 基于DNA纳米花的细胞自噬基因沉默用于增敏抗肿瘤化疗. 高等学校化学学报, 2020, 41(7): 1461.

ZHANG Kaixiang, LIU Junjie, SONG Qiaoli, WANG Danyu, SHI Jinjin, ZHANG Haiyue, LI Jinghong. Multifunctional DNA Nanoflowers for Autophagy Inhibition and Enhanced Antitumor Chemotherapy^†. Chem. J. Chinese Universities, 2020, 41(7): 1461.

图/表 7

Scheme 1 Schematic diagram of DNA nanoflower for autophagy inhibition and enhanced antitumor chemotherapy DNA nanoflowers(NFs) were synthesized by rolling circle amplification(RCA), encoding 3 functional sequences: DNAzyme for ATG5 mRNA, AS1411 aptamer and Dox binding sites(NFs/AS1411/ATG5/DOX). NFs can be efficiently uptaken by cancer cells, degraded by the acidic condition in lysosome and release Mg2+ to trigger the cleavage activity of DNAzyme. The activated DNAzymes would cleave ATG5 mRNA inside cells and inhibit autophagy process, which lead to enhancing antitumor chemotherapy.

Table 1 Sequence of oligonucleotides used in the experiment

Sample	Sequence(5'- 3')
ATG5 DNAzyme 1	CAATCCCAGGCTAGCTACAACGA CCAGAG
ATG5 DNAzyme 2	TGCAATCCCAGGCTAGCTACAACGACCAGAGTTG
ATG5 F	FAM-CCAGAGTTGCTTGTGATCTT
ATG5 Q	CTGTCATTTTGCAATCCCA-BHQ
ATG5 substrate	AAGATCACAAGCAACTCTGG/rA/rU/GGGATTGCAAAATGACAG
Circle	CACAACCACCACCACCAACAACTCTGGTCGTTGTAGCTAGCCTGGGATTGCAAACCACCAC
Primer	GTGGTGGTTGTGGTGGTGGTGGTT
Random circle	CACGCTCACCTCCACCAACATAAATGCAAATAGAAACTAGGCTTTGATAATAAAGGTCCAGGAC
Random primer	GAGGTGAGCGTGGTCCTGGACCTT

Fig.1 Characterization of synthesized DNA nanoflowers (A) Agarose gel electrophoresis of synthesized DNA nanoflower. Lane 1: DNA ladder, lane 2: ligated DNA circle, lane 3: synthesized NFs; (B) DLS analysis of size distribution of NFs; (C) Zeta potential of NFs; (D) SEM image of NFs; (E) TEM imaging of NFs; (F) EDS element analysis; (G) ICP-MS analysis of the released Mg2+ concentration of NFs at pH 7.4, 6.0 and 4.5.

Fig.2 DNAzyme cleavage activity of NFs (A) Fluorescent analysis of NFs and DNAzyme with different molar ratios; (B) cleavage analysis without Mg2+; (C) PAGE analysis of cleavage activity with different pH values and NFs/substrate molar ratios. Left lane: ATG5 substrate. The substrate concentration was constantly 400 nmol/L. Time ranges in (C) were 10, 120 and 300 min, respectively; (D) cleavage activity of DNAzyme with different concentrations of Mg2+.

Fig.3 Dox loading and release capacity of NFs (A) 100 μmol/L Dox was incubated with different concentrations of NFs; (B) pH and DNase triggered release of Dox from NFs; (C) DNAzyme cleavage activity of NFs before and after Dox loading.

Fig.4 Tumor cell targeting ability of NFs (A) Confocal imaging of MCF-7 cells incubated with Dox, NFs/AS1411/ATG5/Dox and Random NFs/Dox; (B) flow cytometry analysis of MCF-7 cells incubated with Dox, NFs/AS1411/ATG5/Dox and Random NFs/Dox.

Fig.5 Cell toxicity and apoptosis induced by NFs (A) RT-PCR analysis of ATG5 expression level in MCF-7 cells incubated with NFs/AS1411/ATG5 or Random NFs. a. Blank control; b. NFs/AS1411/ATG5; c. Random NFs; (B) cytotoxicity test of different NFs incubated with MCF-7 cells for 24 h. a. Dox; b. NFs/AS1411/ATG5/Dox; c. Random NFs/Dox; d. NFs/AS1411/ATG5; Random NFs; (C) flow cytometry quantification of apoptotic Annexin V/PI stained MCF-7 cells induced by different NFs.

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基于DNA纳米花的细胞自噬基因沉默用于增敏抗肿瘤化疗

Multifunctional DNA Nanoflowers for Autophagy Inhibition and Enhanced Antitumor Chemotherapy^†

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基于DNA纳米花的细胞自噬基因沉默用于增敏抗肿瘤化疗

Multifunctional DNA Nanoflowers for Autophagy Inhibition and Enhanced Antitumor Chemotherapy†

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Multifunctional DNA Nanoflowers for Autophagy Inhibition and Enhanced Antitumor Chemotherapy^†