金纳米笼探针用于线粒体成像和光热损伤细胞★

doi:10.6023/A23050222

化学学报 ›› 2023, Vol. 81 ›› Issue (10): 1301-1310.DOI: 10.6023/A23050222 上一篇下一篇

所属专题：庆祝《化学学报》创刊90周年合辑

研究论文

金纳米笼探针用于线粒体成像和光热损伤细胞^★

孙丽^a, 王亚静^a, 李涛^b, 郭英姝^b^,^*(), 张书圣^a^,^*()

a 临沂大学化学化工学院山东临沂 276005
b 齐鲁工业大学(山东省科学院)化学与化工学院济南 250353

投稿日期:2023-05-12 发布日期:2023-07-17
作者简介:
^★庆祝《化学学报》创刊90周年.
基金资助:
国家自然科学基金(22276102); 泰山学者工程(tsqn202211212); 山东省自然科学基金(ZR2023JQ004); 山东省自然科学基金(ZR2022MB024); 济南市引进创新团队(202228027); 山东省高等学校青创科技计划创新团队(2020KJC003); 齐鲁工业大学(山东省科学院)科教产融合试点工程项目(2023PYI002)

Au Nanocages Probes for Mitochondrial Imaging and Photothermal Damage Cells^★

Li Sun^a, Yajing Wang^a, Tao Li^b, Yingshu Guo^b(), Shusheng Zhang^a()

a School of Chemistry and Chemical Engineering, Linyi University, Linyi, Shandong 276005
b School of Chemistry and Chemical Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353

Received:2023-05-12 Published:2023-07-17
Contact: *E-mail: yingshug@126.com; shushzhang@126.com
About author:
^★Dedicated to the 90th anniversary of Acta Chimica Sinica.
Supported by:
National Natural Science Foundation of China(22276102); Young Taishan Scholar Program of Shandong Province of China(tsqn202211212); Shandong Provincial Natural Science Foundation(ZR2023JQ004); Shandong Provincial Natural Science Foundation(ZR2022MB024); University Institute Innovation Team of Jinan(202228027); Development Plan of Youth Innovation Team in Colleges and Universities of Shandong Province(2020KJC003); Science, Education and Industry Integration Pilot Project Plan of Qilu University of Technology (Shandong Academy of Sciences)(2023PYI002)

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1. Supporting information-A23050222.pdf(940KB)

摘要/Abstract

线粒体是许多细胞行为的关键调节细胞器, 线粒体膜电位降低被认为是细胞凋亡所发生的最早事件之一, 因此线粒体成像及其膜电位的检测分析, 对疾病的检测与治疗有重要的科学意义. 采用金纳米笼(Au nanocages, AuNCs)介导的光热损伤与温度敏感的药物释放相结合, 开发了一种线粒体靶向的荧光纳米探针AuNCs/PLEL/JC/KLA. 引入一种线粒体靶向肽(KLAKLAKKLAKLAK, KLA), 作为纳米探针的“指向标”, 指引着探针特异地靶向到细胞线粒体部位, 随后在近红外光的照射下, AuNCs吸收光能转化为热量, 实现光热介导的细胞损伤. 同时, 高温促使外层温敏水凝胶发生凝胶-溶胶转变, 实现荧光染料(JC-10)的释放. 所释放的JC-10荧光染料可根据线粒体的活力表现出两种荧光信号, 用于监测线粒体膜电位的变化. 总之, 该荧光纳米探针不仅实现了线粒体靶向的荧光成像与损伤细胞, 同时还可以监测线粒体膜电位的变化.

关键词: 线粒体, 荧光探针, 光热损伤, 荧光成像, 膜电位

Mitochondria are the key regulatory organelles of many cell behaviors, and the reduction of mitochondrial membrane potential is considered to be one of the earliest events of cell apoptosis. Therefore, mitochondrial imaging and the detection and analysis of mitochondrial membrane potential are of great scientific significance for the detection and treatment of diseases. In this work, AuNCs/PLEL/JC/KLA, a mitochondrial targeted fluorescent nanoprobe, was developed using Au nanocages (AuNCs) mediated photothermal damage combined with temperature-sensitive drug release. At the same time, the temperature-sensitive hydrogel poly(d,l-lactide)-poly(ethylene glycol)-poly(d,l-lactide) (PDLLA-PEG-PDLLA, PLEL) is used as the outer structure to control the release. A mitochondrial targeting peptide (KLAKLAKKLAKLAK, KLA) was introduced as the "pointer" of the nanoprobe to specifically target the mitochondria. The colocalization experiment showed that the nanoprobe was highly colocalized with mitochondria, indicating that the nanoprobe was selectively enriched in mitochondria. It is worth noting that the nanoprobe has excellent photothermal properties, and its photothermal conversion efficiency can be as high as 39.11%. Therefore, under the irradiation of near infrared light, the probe can absorb light energy into heat. Subsequently, the results of Cell Counting Kit 8 (CCK-8) confirmed that the nanoprobes could achieve local photothermal damage at mitochondrial sites, triggering high temperature mediated mitochondrial dysfunction and inducing apoptosis of cancer cells. Meanwhile, rheological analysis and fluorescence curve showed that high temperature promotes the gel-sol transformation of PLEL thermosensitive hydrogel and realizes the release of fluorescent dye (JC-10). The confocal images of the cells showed that the released JC-10 fluorescent dye can display red and green fluorescence signals based on mitochondrial activity. In conclusion, the fluorescence nanoprobe can not only achieve mitochondrial targeted fluorescence imaging and damage cells, but also monitor the changes of mitochondrial membrane potential.

Key words: mitochondrion, fluorescent probe, photothermal damage, fluorescence imaging, membrane potential

引用此文

孙丽, 王亚静, 李涛, 郭英姝, 张书圣. 金纳米笼探针用于线粒体成像和光热损伤细胞^★[J]. 化学学报, 2023, 81(10): 1301-1310.

Li Sun, Yajing Wang, Tao Li, Yingshu Guo, Shusheng Zhang. Au Nanocages Probes for Mitochondrial Imaging and Photothermal Damage Cells^★[J]. Acta Chimica Sinica, 2023, 81(10): 1301-1310.

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图/表 11

图1. AuNCs/PLEL/JC/KLA纳米探针的合成思路以及靶向线粒体光热损伤的原理图

Figure 1. Synthesis of AuNCs/PLEL/JC/KLA nanoprobes and schematic diagram of targeting mitochondria photothermal damage

图2. TEM图: (a) AgNPs, (b) AuNCs, (c) PLEL, (d) AuNCs/PLEL/JC/KLA

Figure 2. TEM images of (a) AgNPs, (b) AuNCs, (c) PLEL, (d) AuNCs/PLEL/JC/KLA

图3. (a)粒径图: AgNPs, AuNCs, PLEL, AuNCs/PLEL/JC和AuNCs/PLEL/JC/KLA. (b) Zeta电位图: AuNCs, PLEL, AuNCs/PLEL, JC, AuNCs/PLEL/JC, KLA和AuNCs/PLEL/JC/KLA

Figure 3. (a) Particle size graphs of AgNPs, AuNCs, PLEL, AuNCs/ PLEL/JC and AuNCs/PLEL/JC/KLA. (b) Zeta potentials of AuNCs, PLEL, AuNCs/PLEL, JC, AuNCs/PLEL/JC, KLA and AuNCs/PLEL/ JC/KLA

图4. (a)紫外-可见吸收光谱图: AgNPs, AuNCs, JC和AuNCs/PLEL/ JC; (b) FTIR图: PLEL和AuNCs/PLEL/JC/KLA

Figure 4. (a) UV-vis curves of AgNPs, AuNCs, JC and AuNCs/ PLEL/JC; (b) FTIR curves of PLEL and AuNCs/PLEL/JC/KLA

图5. (a)不同浓度的AuNCs溶液在1.5 W/cm2的NIR光照射10 min的升温曲线; (b) H2O, AuNCs, AuNCs/PLEL和AuNCs/PLEL/JC/KLA在1.5 W/cm2的NIR光照射10 min的升温曲线; (c) AuNCs/PLEL/ JC/KLA在3次近红外光开-关周期内的加热-冷却曲线; (d) AuNCs/ PLEL/JC/KLA在1.5 W/cm2的NIR光照射10 min的红外热成像图

Figure 5. (a) Temperature rise curves of different concentrations of AuNCs solution irradiated with 1.5 W/cm2 NIR light for 10 min; (b) temperature rise curves of H2O, AuNCs, AuNCs/PLEL and AuNCs/ PLEL/JC/KLA under NIR irradiation of 1.5 W/cm2 for 10 minutes; (c) Heating and cooling curves of AuNCs/PLEL/JC/KLA during three near-infrared on-off cycles; (d) Infrared thermogram of AuNCs/ PLEL/JC/KLA at 1.5 W/cm2 NIR irradiation for 10 min

图6. 流变学分析图: (a) PLEL和(b) AuNCs/PLEL/JC/KLA

Figure 6. Rheological analysis diagrams of (a) PLEL and (b) AuNCs/ PLEL/JC/KLA

图7. AuNCs/PLEL/JC/KLA在37和40 ℃时上清液中JC-10的荧光光谱图

Figure 7. Fluorescence spectra of JC-10 in AuNCs/PLEL/JC/KLA supernatant at 37 and 40 ℃

图8. 细胞活力图: (a)在无NIR激光的照射下, 用不同浓度的AuNCs、AuNCs/PLEL/JC和AuNCs/PLEL/JC/KLA处理Ramos细胞; (b)在NIR激光的照射下, 用不同浓度的AuNCs、AuNCs/PLEL/JC和AuNCs/ PLEL/JC/KLA处理Ramos细胞

Figure 8. Cell viability diagrams of (a) Ramos cells treated with different concentrations of AuNCs, AuNCs/PLEL/JC and AuNCs/PLEL/ JC/KLA without NIR laser irradiation; (b) Ramos cells was treated with different concentrations of AuNCs, AuNCs/PLEL/JC and AuNCs/PLEL/ JC/KLA under the irradiation of NIR laser

图9. Ramos细胞分别与JC-10和AuNCs/PLEL/JC/KLA探针孵育后的共定位图像, 比例尺为50 μm

Figure 9. Colocalization images of Ramos cells incubated with JC-10 and AuNCs/PLEL/JC/KLA probes respectively, with a scale of 50 μm

图10. 探针与Ramos细胞孵育后, 用NIR光照射不同时间的细胞共聚焦成像图, 比例尺为10 μm

Figure 10. After incubating the probe with Ramos cells, confocal images of the cells were irradiated with NIR light for different times, with a scale of 10 μm

图11. 经过不同浓度的羰基氰化氯苯腙(CCCP)处理后的Ramos细胞与AuNCs/PLEL/JC/KLA孵育的共聚焦图像, 比例尺为50 μm

Figure 11. Confocal image of Ramos cells incubated with AuNCs/PLEL/JC/KLA after being treated with different concentrations of carbonyl chlorophenylhydrazone cyanide (CCCP), with a scale of 50 μm

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金纳米笼探针用于线粒体成像和光热损伤细胞^★

Au Nanocages Probes for Mitochondrial Imaging and Photothermal Damage Cells^★

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金纳米笼探针用于线粒体成像和光热损伤细胞★

Au Nanocages Probes for Mitochondrial Imaging and Photothermal Damage Cells★

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金纳米笼探针用于线粒体成像和光热损伤细胞^★

Au Nanocages Probes for Mitochondrial Imaging and Photothermal Damage Cells^★