Direct Fluorescence Monitoring of the Delivery and Cellular Uptake of a Cancer-Targeted RGD Peptide-Appended Naphthalimide Theragnostic Prodrug
Direct Fluorescence Monitoring of the Delivery and Cellular Uptake of a Cancer-Targeted RGD Peptide-Appended Naphthalimide Theragnostic Prodrug
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DOI：
10.1021/ja303998y
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发表时间：
2012-08-01
影响因子：
15
通讯作者：
Kim, Jong Seung
中科院分区：
文献类型：
--
作者：
Lee, Min Hee;Kim, Jin Young;Kim, Jong Seung
本文介绍的是一种多组分合成策略，其允许直接基于荧光监测靶向细胞摄取和缀合治疗剂的释放。具体而言，我们在这里报告的设计，合成，光谱表征，并在体外初步生物学评价的RGD肽附加的萘酰亚胺前CPT（化合物1）。化合物1是由作为可裂解接头的二硫键、作为荧光报告分子的萘二甲酰亚胺部分、作为癌症靶向单元的RGD环肽和作为模型活性剂的喜树碱（CPT）组成的多功能分子。在pH 7.4的水性介质中与游离硫醇反应后，发生二硫化物裂解。这导致游离CPT活性剂的释放，以及产生红移荧光发射（λ（max）= 535 nm）。共聚焦显微镜实验表明，1是由U87细胞比C6细胞优先采取。在竞争实验的基础上，涉及冈田酸，内吞作用的抑制剂，它的结论是摄取发生通过RGD依赖的内吞机制。在U87细胞中，活性CPT有效载荷在内质网内释放，如使用已知内质网选择性染料的基于荧光的共定位研究所推断的。本药物递送系统（DDS）可以代表所谓的治疗诊断剂开发的新方法，其中产生治疗效果和药物摄取相关的成像信息，并且可以在亚细胞水平上容易地监测。在适当的时候，结合物1中体现的策略可以允许更精确地监测剂量水平，以及改善对细胞摄取和释放机制的理解。
Presented here is a multicomponent synthetic strategy that allows for the direct, fluorescence-based monitoring of the targeted cellular uptake and release of a conjugated therapeutic agent. Specifically, we report here the design, synthesis, spectroscopic characterization, and preliminary in vitro biological evaluation of a RGD peptide-appended naphthalimide pro-CPT (compound 1). Compound 1 is a multifunctional molecule composed of a disulfide bond as a cleavable linker, a naphthalimide moiety as a fluorescent reporter, an RGD cyclic peptide as a cancer-targeting unit, and camptothecin (CPT) as a model active agent. Upon reaction with free thiols in aqueous media at pH 7.4, disulfide cleavage occurs. This leads to release of the free CPT active agent, as well as the production of a red-shifted fluorescence emission (lambda(max) = 535 nm). Confocal microscopic experiments reveal that 1 is preferentially taken up by U87 cells over C6 cells. On the basis of competition experiments involving okadaic acid, an inhibitor of endocytosis, it is concluded that uptake takes place via RGD-dependent endocytosis mechanisms. In U87 cells, the active CPT payload is released within the endoplasmic reticulum, as inferred from fluorescence-based colocalization studies using a known endoplasmic reticulum-selective dye. The present drug delivery system (DDS) could represent a new approach to so-called theragnostic agent development, wherein both a therapeutic effect and drug uptake-related imaging information are produced and can be readily monitored at the subcellular level. In due course, the strategy embodied in conjugate 1 could allow for more precise monitoring of dosage levels, as well as an improved understanding of cellular uptake and release mechanisms.