Muddana Hari S, Morgan Thomas T, Adair James H, Butler Peter J
Department of Bioengineering, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Nano Lett. 2009 Apr;9(4):1559-66. doi: 10.1021/nl803658w.
Progress toward clinical application of biodegradable fluorescent calcium phosphate (CP) nanoparticles as a bioimaging agent requires detailed knowledge of chromophore interaction with CP. As readouts of this cargo-matrix interaction, we determined the principle photophysical properties of Cy3 encapsulated in CP nanparticles (CPNPs) using steady-state and time-resolved fluorescence spectroscopy. Fluorescence correlation spectroscopy (FCS)-determined diffusion coefficients and associated hydrodynamic radii confirmed the presence of highly monodisperse CPNPs with radii ranging from 7 to 10 nm. Single CP nanoparticles were 20 times brighter than free dye molecules because of a CP-induced 5-fold increase in quantum efficiency and encapsulation of four dye molecules per particle. Solvatochromic shifts resulting from hydrogen bonding between free dye and solvent or restricted intramolecular mobility by solvent viscosity were absent when Cy3 was encapsulated in CP. Encapsulation-mediated increases in radiative decay rates and decreases in nonradiative decay rates resulting in longer fluorescence lifetimes of Cy3 were attributed to solvent and CP-related local refractive indices and restricted flexibility of dye by rigid CP. Enhanced brightness of CPNPs enabled imaging of single nanoparticles under epifluorescence using both standard and total internal reflection fluorescence (TIRF) modes with camera exposure times on the order of tens of milliseconds. These enhanced photophysical properties together with excellent biocompatibility make CPNPs ideal for bioimaging applications ranging from single-molecule tracking to in vivo tumor detection and offer the possibility of timed codelivery of drugs to control cell function.
将可生物降解的荧光磷酸钙（CP）纳米颗粒作为生物成像剂应用于临床，需要详细了解发色团与CP的相互作用。作为这种载药-基质相互作用的读数，我们使用稳态和时间分辨荧光光谱法测定了包裹在CP纳米颗粒（CPNP）中的Cy3的主要光物理性质。荧光相关光谱（FCS）测定的扩散系数和相关的流体动力学半径证实了存在高度单分散的CPNP，其半径范围为7至10 nm。单个CP纳米颗粒比游离染料分子亮20倍，这是因为CP使量子效率提高了5倍，且每个颗粒包裹了四个染料分子。当Cy3包裹在CP中时，不存在由游离染料与溶剂之间的氢键或溶剂粘度导致的分子内迁移受限所引起的溶剂化显色位移。包裹介导的辐射衰减率增加和非辐射衰减率降低导致Cy3的荧光寿命延长，这归因于溶剂和CP相关的局部折射率以及刚性CP对染料灵活性的限制。CPNP增强的亮度使得在落射荧光下使用标准和全内反射荧光（TIRF）模式，通过几十毫秒量级的相机曝光时间对单个纳米颗粒进行成像成为可能。这些增强的光物理性质以及出色的生物相容性使CPNP成为从单分子追踪到体内肿瘤检测等生物成像应用的理想选择，并提供了定时共递送药物以控制细胞功能的可能性。
