Britton Chance Center for Biomedical Photonics at, Wuhan National Laboratory for Optoelectronics-Hubei, Bioinformatics & Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology Wuhan 430074, PR China.
Small. 2013 Dec 20;9(24):4183-93. doi: 10.1002/smll.201300994. Epub 2013 Jul 5.
Based on the multivalent binding capability of streptavidin (SA) to biotin, a multifunctional quantum dot probe (QD-(AS-ODN+p160)) coupled with antisense oligonucleotide (AS-ODN) and peptide p160 is designed for real-time tracking of targeted delivery of AS-ODN and regulation of folate receptor-α (hFR-α) in MCF-7 breast cancer cells. Fluorescence spectra, capillary electrophoresis (CE) and dynamic light scattering (DLS) are used to characterize the conjugation of AS-ODN and p160 with quantum dots (QDs), DLS results confirm the well stability of the probe in aqueous media. Confocal imaging and quantitative flow cytometry show that QD-(AS-ODN+p160) is able to specifically target human breast cancer MCF-7 cells. Low temperature and ATP depletion treatments reveal the cellular uptake of QD-(AS-ODN+p160) is energy-dependent, and the effects of inhibition agents and co-localization imaging further confirm the endocytic pathway is mainly receptor-mediated. Transmission electron microscopy (TEM) shows the intracellular delivery and endosomal escape of QD probe along with incubation time extended. Two transfection concentrations of QD probe (10 nM and 50 nM) below half inhibitory concentration (IC50 ) value are chosen according to MTT assay. Real-time PCR shows at these two concentration cases the relative mRNA expression levels of hFR-α reduce to 72.5 ± 3.9% and 17.6 ± 1.0%, respectively. However, western blot and quantitative ELISA analysis show the expression level of hFR-α protein has a significant decrease only at 50 nM, indicating that gene silence is concentration-dependent. These results demonstrate that the QD-(AS-ODN+p160) probe not only achieves gene silence in a cell-specific manner but also achieves real-time tracking during AS-ODN intracellular delivery.
基于链霉亲和素（SA）对生物素的多价结合能力，设计了一种多功能量子点探针（QD-（AS-ODN+p160）），该探针与反义寡核苷酸（AS-ODN）和肽 p160 偶联，用于实时跟踪靶向递送 AS-ODN 并调节叶酸受体-α（hFR-α）在 MCF-7 乳腺癌细胞中的作用。荧光光谱、毛细管电泳（CE）和动态光散射（DLS）用于表征 AS-ODN 和 p160 与量子点（QDs）的缀合，DLS 结果证实探针在水介质中具有良好的稳定性。共聚焦成像和定量流式细胞术显示，QD-（AS-ODN+p160）能够特异性靶向人乳腺癌 MCF-7 细胞。低温和 ATP 耗尽处理表明 QD-（AS-ODN+p160）的细胞摄取是能量依赖性的，抑制剂和共定位成像的作用进一步证实了内吞途径主要是受体介导的。透射电子显微镜（TEM）显示随着孵育时间的延长，QD 探针在细胞内的递送和内体逃逸。根据 MTT 测定，选择低于半抑制浓度（IC50）值的两个 QD 探针转染浓度（10 nM 和 50 nM）。实时 PCR 显示，在这两种浓度下，hFR-α 的相对 mRNA 表达水平分别降低至 72.5±3.9%和 17.6±1.0%。然而，Western blot 和定量 ELISA 分析表明，只有在 50 nM 时 hFR-α 蛋白的表达水平才显著下降，表明基因沉默是浓度依赖性的。这些结果表明，QD-（AS-ODN+p160）探针不仅以细胞特异性方式实现基因沉默，而且在 AS-ODN 细胞内递送过程中实现实时跟踪。
