Department of Biomedical Engineering, Characterization Facility, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Mol Pharm. 2011 Feb 7;8(1):176-84. doi: 10.1021/mp1002587. Epub 2010 Dec 3.
This work demonstrates the use of confocal Raman microscopy (CRM) to measure the dynamics of cellular uptake and localization of gold nanoparticles (GNP) with nanoscale resolution. This is important as nanoparticle cellular interactions are increasingly under investigation to support applications as diverse as drug delivery, gene transfection and a variety of heat and radiation based therapeutics. At the heart of these applications is a need to know the dynamics of nanoparticle cellular uptake and localization (i.e., cell membrane, cytoplasm or nucleus). This process can change dramatically based on size, charge, shape and ligand attached to the nanoparticle. While electron microscopy, atomic emission spectroscopy and histology can be used to assess cellular uptake, they are labor intensive and post-mortem and can miss critical dynamics of the process. For this reason investigators are increasingly turning to optically active nanoparticles that allow direct microscopic interrogation of uptake. Here we show that CRM adds to this evolving armamentarium as a fast, noninvasive, and label-free technique to dynamically study cellular uptake of GNPs with subcellular detail in cancer. Raman laser interaction with GNPs inside cells shows unique spectroscopic features corresponding to the intracellular localization of GNPs over 2 to 24 h at the membrane, cytoplasm or nucleus that are separately verified by histology (silver staining) and electron microscopy. These results show that CRM has the potential to facilitate high-throughput study of the dynamics and localization of a variety of GNPs in multiple cell types.
这项工作展示了如何使用共聚焦拉曼显微镜（CRM）以纳米级分辨率测量金纳米粒子（GNP）的细胞摄取和定位动力学。这一点很重要，因为人们越来越多地研究纳米颗粒与细胞的相互作用，以支持各种应用，如药物输送、基因转染以及各种基于热和辐射的疗法。这些应用的核心是需要了解纳米颗粒的细胞摄取和定位（即细胞膜、细胞质或细胞核）动力学。这一过程会根据纳米颗粒的尺寸、电荷、形状和连接的配体而发生显著变化。尽管电子显微镜、原子发射光谱学和组织学可用于评估细胞摄取，但这些方法劳动强度大且为死后检测，可能会错过该过程的关键动态。出于这个原因，研究人员越来越倾向于使用允许直接微观检测摄取的光学活性纳米颗粒。在这里，我们表明 CRM 作为一种快速、非侵入性且无需标记的技术，可用于在癌症中以亚细胞细节动态研究 GNP 的细胞摄取，从而为这一不断发展的技术手段增添了新的力量。拉曼激光与细胞内 GNP 的相互作用显示出与 GNP 细胞内定位相对应的独特光谱特征，在 2 至 24 小时内可在细胞膜、细胞质或细胞核中分别得到组织学（银染）和电子显微镜的验证。这些结果表明，CRM 有可能促进对多种细胞类型中各种 GNP 的动力学和定位的高通量研究。
