Intracellular delivery and targeting of nanoparticles

描述(由申请人提供) 摘要：纳米粒子具有重要的生物医学应用，从基因治疗人类疾病到利用荧光探针了解细胞的基本功能。现在已经有可能合成几乎任何性质或大小的纳米粒子，将它们定向到特定的细胞，并将它们功能化以靶向细胞内的位置，但将纳米粒子穿过质膜运送到他们的目标仍然是一个挑战。我们最近展示了第一种非侵入性地将半导体纳米颗粒(称为量子点)同时输送到多个细胞的细胞质中的方法。给药需要丁酸吡喃与阳离子多肽的结合，以使QD与质膜直接相互作用。量子点穿过质膜的能力为将其他纳米颗粒输送到活细胞提供了令人兴奋的可能性。这项研究计划的第一个目标是确定丙二醇丁酸酯介导的递送的分子和细胞机制，并将其扩展到其他纳米颗粒，最终目标是用所选择的纳米颗粒靶向特定的细胞内位置。新的成像方法，包括单粒子跟踪荧光显微镜，将被用来探测纳米粒子的运动以及它在穿过质膜和细胞质时与丙二醇丁酸酯的相互作用。由于丙二醇丁酸酯介导的递送可能并不适合所有的纳米颗粒和所有的应用，我们还将致力于开发一套基于充分表征的内体摄取途径的胞液递送和靶向方法。这两种给药方法都将与细胞对纳米颗粒的反应研究结合起来进行，目的是优化给药并将干扰降至最低。在这项研究过程中，足够明亮的量子点将被用来探索细胞运输的基本问题，包括在拥挤的细胞环境中的扩散，囊泡介导的运输，以及核靶向。 公共卫生相关性：这项研究解决了利用纳米技术造福人类健康的一个根本挑战：将纳米颗粒输送到活细胞中。两种递送方法，移位和内吞，将结合细胞反应的研究来探索，以获得最佳递送和最小的干扰。这些研究将使用模块化的功能化方案，可以扩展到各种各样的纳米颗粒，在许多不同的学科中产生广泛的影响，包括癌症治疗、基因传递和细胞成像。

DESCRIPTION (Provided by the applicant) Abstract: Nanoparticles have important biomedical applications ranging from the treatment of human disease with gene therapy to understanding basic cellular functions with fluorescent probes. It is now possible to synthesize nanoparticles with nearly any property or size, direct them to specific cells, and functionalize them to target intracellular locations, but delivering nanoparticles across the plasma membrane to reach their targets remains a challenge. We have recently demonstrated the first method for non-invasive delivery of semiconductor nanoparticles, known as quantum dots (QDs), to the cytosol of multiple cells simultaneously. Delivery requires pyrenebutyrate in combination with a cationic peptide for direct interaction of the QD with the plasma membrane. The ability of QDs to cross the plasma membrane offers exciting possibilities for the delivery of other nanoparticles to living cells. The first goal of this research program is to determine the molecular and cellular mechanism of pyrenebutyrate-mediated delivery and extend it to other nanoparticles with the ultimate goal of targeting specific intracellular sites with nanoparticles of choice. Novel imaging methods, including single particle tracking fluorescence microscopy, will be used to probe the motion of the nanoparticle and its interaction with pyrenebutyrate as it moves across the plasma membrane and through the cytosol. As pyrenebutyrate-mediated delivery may not be suitable for all nanoparticles and all applications, we will also work to develop a suite of cytosolic delivery and targeting methods that are based on the well-characterized endosomal uptake pathways. Both delivery methods will be carried out in conjunction with studies of cellular response to nanoparticles that aim to optimize delivery and minimize disruption. In the course of this research, QDs, which are sufficiently bright for imaging at the single particle level, will be used to probe fundamental questions of cellular transport including diffusion through the crowded cellular environment, vesicle-mediated transport, and nuclear targeting. Public Health Relevance: This research addresses a fundamental challenge in the use of nanotechnology for the benefit of human health; the delivery of nanoparticles into living cells. Two delivery methods, translocation and endocytosis, will be explored in conjunction with studies of cellular response for optimal delivery and minimal disruption. These studies will use a modular scheme for functionalization that can be extended to a wide variety of nanoparticles for broad impact in many different disciplines including cancer therapy, gene delivery, and cellular imaging.