Division of Bioengineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117574.
Langmuir. 2010 May 4;26(9):6689-94. doi: 10.1021/la904011q.
Investigation of the intracellular fate of small interference RNA (siRNA), after their delivery into cells by nanoparticles, is of great interest to the development of more efficient methods for transfection of siRNA. The fluorescence resonance energy transfer (FRET) based method using upconversion fluorescent nanoparticles (UCN) as energy donor is established to study intracellular release and biostability of siRNA in live cells. The UCN/siRNA-BOBO3 complex is prepared where BOBO-3-stained siRNAs are attached to the surface of amino-group-modified silica/NaYF(4):Yb,Er UCN. The energy is transferred from the UCN donor to the BOBO-3 acceptor under excitation of a near-infrared (NIR) laser. The FRET efficiency is established as a reliable parameter to follow the release and biostability of siRNA in phosphate buffered saline (PBS) and live cells. Intracellular FRET analysis shows that siRNA is gradually released into cells for a duration of 24 h, which is confirmed by confocal microscopy colocalization measurements. The application of this straightforward and sensitive upconversion FRET technique can gain real-time information on intracellular fate of siRNA and provide a bright outlook for in vitro and even in vivo detection of biological molecules.
研究小分子干扰 RNA（siRNA）在纳米颗粒介导的细胞内递送后，其细胞内命运对于开发更有效的 siRNA 转染方法非常重要。本研究建立了基于荧光共振能量转移（FRET）的方法，使用上转换荧光纳米粒子（UCN）作为能量供体，研究 siRNA 在活细胞内的释放和生物稳定性。其中，UCN/siRNA-BOBO3 复合物是通过将 BOBO-3 标记的 siRNA 附着在氨基修饰的硅/NaYF(4):Yb,Er UCN 表面来制备的。在近红外（NIR）激光激发下，能量从 UCN 供体转移到 BOBO-3 受体。FRET 效率可作为一种可靠的参数，用于跟踪 siRNA 在磷酸盐缓冲盐水（PBS）和活细胞中的释放和生物稳定性。细胞内 FRET 分析表明，siRNA 逐渐释放到细胞中持续 24 小时，这通过共聚焦显微镜共定位测量得到了证实。这种简单灵敏的上转换 FRET 技术的应用可以实时获取 siRNA 细胞内命运的信息，为体外甚至体内生物分子的检测提供了广阔的前景。
