David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA.
Integr Biol (Camb). 2013 Jan;5(1):224-30. doi: 10.1039/c2ib20155k.
One important barrier facing the delivery of short interfering RNAs (siRNAs) via synthetic nanoparticles is the rate of nanoparticle disassembly. However, our ability to optimize the release kinetics of siRNAs from nanoparticles for maximum efficacy is limited by the lack of methods to track their intracellular disassembly. Towards this end, we describe the design of two different siRNA-based fluorescent probes whose fluorescence emission changes in response to the assembly state of the nanoparticle. The first probe design involves a redox-sensitive fluorescence-quenched probe that fluoresces only when the nanoparticle is disassembled in a reductive environment. The second probe design is based on a FRET-labeled siRNA pair that fluoresces due to the proximity of the siRNA pair when the nanoparticle is intact. In both approaches, the delivery vehicle need not be labeled. The utility of these probes was investigated with a lipidoid nanoparticle (LNP) as proof-of-concept in both extracellular and intracellular environments. Fluorescence kinetic data from both probes were fit to a two-phase release and decay curve and subsequently quantified to give intracellular disassembly rate constants. Quantitative analysis revealed that the rate constant of siRNA release measured via the fluorescence-quenched probe was almost identical to the rate constant for nanoparticle disassembly measured via the FRET-labeled probes. Furthermore, these probes were utilized to determine subcellular localization of LNPs with the use of automated high-resolution microscopy as they undergo dissociation. Interestingly, this work shows that large amounts of siRNA remain inside vesicular compartments. Altogether, we have developed new siRNA probes that can be utilized with multiple nanocarriers for quantitative and qualitative analysis of nanoparticle dissociation that may serve as a design tool for future delivery systems.
一种重要的障碍是通过合成纳米粒子传递短干扰 RNA(siRNAs) 的速度纳米粒子的解体。然而，我们优化 siRNA 从纳米粒子中释放动力学以达到最大疗效的能力受到缺乏方法的限制来跟踪它们的细胞内解体。为此，我们描述了两种不同的基于 siRNA 的荧光探针的设计，其荧光发射响应纳米粒子的组装状态而变化。第一种探针设计涉及一种氧化还原敏感的荧光猝灭探针，只有在纳米粒子在还原环境中解组装时才会发光。第二种探针设计基于 FRET 标记的 siRNA 对，当纳米粒子完整时，由于 siRNA 对的接近而发光。在这两种方法中，输送载体不需要标记。我们用脂质体纳米颗粒 (LNP) 作为概念验证，在细胞外和细胞内环境中研究了这些探针的实用性。来自两种探针的荧光动力学数据拟合到两相释放和衰减曲线，并随后进行定量分析以给出细胞内解组装速率常数。定量分析表明，通过荧光猝灭探针测量的 siRNA 释放速率常数与通过 FRET 标记探针测量的纳米粒子解组装速率常数几乎相同。此外，这些探针还用于通过自动高分辨率显微镜确定 LNPs 的亚细胞定位，因为它们发生解离。有趣的是，这项工作表明大量的 siRNA 仍然留在囊泡隔间内。总之，我们已经开发了新的 siRNA 探针，可与多种纳米载体一起用于定量和定性分析纳米粒子的解离，这可能成为未来输送系统的设计工具。
