Live-cell-permeable poly (p-phenylene ethynylene)
Live-cell-permeable poly (p-phenylene ethynylene)
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DOI：
10.1002/anie.200701991
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发表时间：
2007-01-01
影响因子：
16.6
通讯作者：
Hancock, Lawrence E.
中科院分区：
文献类型：
--
作者：
Moon, Joong Ho;McDaniel, William;Hancock, Lawrence E.
荧光标记和检测活细胞中的目标生物分子是研究复杂和动态细胞过程的重要方法。[1]许多荧光染料[2]和工程荧光蛋白[3]由于其小尺寸和生物相容性而广泛用于这些应用。然而，这些探针的光稳定性差，限制了它们在长期监测高灵敏度活细胞中的广泛适用性。量子点具有光稳定性好、发光范围窄、亮度高等优点，被认为是一种理想的探针材料。[4]然而，量子点的固有毒性（主要来自重金属核，如二价镉离子）引起了对细胞事件长期监测的关注。[5，6]此外，与QD的表面改性相关的困难也阻碍了它们在活细胞系统中的应用。因此，对克服活细胞成像中的稳定性和毒性问题的新型材料的需求很高。[7-10]共轭聚合物（CP）是一种具有吸引力的材料，可满足荧光显微成像的光学要求。[11]CP具有高的荧光量子产率、大的消光系数和高效的光信号转导。此外，CP的合成多功能性允许广泛选择用于连接生物分子的官能团和偶联化学。尽管CP具有这些良好的性质，但π-共轭芳香骨架的固有疏水性限制了CP在生物体系中的潜在应用。通过在CP侧链中引入带电官能团，已经证明了体外核酸，[12]蛋白质，[13，14]细菌[15]或癌细胞[16]的检测。在此，我们介绍荧光共轭聚合物纳米粒子（CPNs），能够荧光成像的活细胞。CPN是通过在CP溶液中的简单溶剂交换制造的稳定的纳米尺寸的荧光颗粒。设计了一种含胺的聚（对苯乙炔）（PPE），并将其制成CPN水溶液，以展示活细胞成像。CPN是细胞可渗透的，仅在细胞溶质中积累，对细胞活力没有任何可测量的抑制。此外，CPN表现出高的耐光漂白性，与市售染料相反。在DMSO和吗啉（1：1v/v）的混合溶剂中，通过钯/铜催化的交叉偶联反应合成了一种PPE作为代表性的CP。我们设计了单体以通过在环氧乙烷接头的末端引入胺基来最小化芳香族主链在水性介质中的π-堆叠（方案1）。PPE中的伯胺基团是特别令人感兴趣的，因为它们在质子化后增加PPE的水溶性，并提供用于偶联生物活性分子的位点。此外，由于减少了链-链相互作用，胺基的质子化增加了荧光强度。[17我们报道了PPE在不良溶剂中的相转化沉淀允许形成稳定的颗粒。[19]PPE在水相中形成各种尺寸的颗粒，这取决于PPE浓度和盐度。在此，连续超滤与乙酸，乙二胺四乙酸（EDTA），和水用于制造稳定的纳米尺寸的颗粒。乙酸辅助去除金属离子污染物（钯和铜），并通过产生排斥力减少CPN中的PPE聚集。荧光光谱中的特征，如加宽的发射，表明颗粒的形成是由聚集驱动的。
Fluorescent labeling and detection of target biological molecules in live cells is an essential way of studying complex and dynamic cellular processes.[1] Many fluorescent dyes [2] and engineered fluorescent proteins [3] are widely used for these applications because of their small size and biocompatibility. However, poor photostability of these probes limits their broad applicability in long-term monitoring of live cells with high sensitivity. Quantum dots (QDs) are considered an alternative probe owing to their excellent optical properties, such as high photostability, narrow emission, and high brightness.[4] However, the inherent toxicity of QDs (mainly from a heavy-metal core, such as divalent cadmium ions) causes concern in long-term monitoring of cellular events.[5, 6] In addition, difficulties associated with surface modification of QDs also retard their applications in live-cell systems. Therefore, novel materials that overcome the stability and toxicity issues in live-cell imaging are in high demand.[7–10] Conjugated polymers (CPs) are attractive materials that meet the optical requirements suitable for fluorescence microscopic imaging.[11] CPs exhibit high fluorescence quantum yield, large extinction coefficients, and efficient optical signal transduction. In addition, the synthetic versatility of CPs allows a wide selection of functional groups and coupling chemistries for attachment of biological molecules. In spite of these promising properties, intrinsic hydrophobicity originating from the π-conjugated aromatic backbone limits the potential applications of CPs in biological systems. By introducing charged functional groups in the CPs side chains, the detection of nucleic acids,[12] proteins,[13, 14] bacteria,[15] or cancer cells [16] in vitro has been demonstrated. Herein, we introduce fluorescent conjugated polymer nanoparticles (CPNs) that are capable of fluorescence imaging of live cells. CPNs are stable, nanometer-sized fluorescent particles fabricated by a simple solvent exchange in a CP solution. An amine containing poly (p-phenylene ethynylene)(PPE) was designed and fabricated into CPN in water to demonstrate live-cell imaging. The CPNs are cell permeable and accumulate exclusively in the cytosol without any measurable inhibition of cell viability. In addition, CPNs exhibit high resistance to photobleaching, in contrast to commercially available dyes. A PPE was synthesized as a representative CP by the palladium/copper-catalyzed cross-coupling reaction in a mixed solvent of DMSO and morpholine (1: 1v/v). We designed monomers to minimize π-stacking of aromatic backbones in aqueous media by introducing amine groups at the end of ethylene oxide linkers (Scheme 1). Primary amine groups in the PPE are of particular interest because they increase aqueous solubility of PPE upon protonation and provide a site for coupling of biologically active molecules. Moreover, protonation of the amine group increases fluorescence intensity because of reduced chain–chain interactions.[17, 18]We reported that the phase-inversion precipitation of PPE in a poor solvent allowed formation of stable particles.[19] PPE formed variously sized particles in aqueous phases, depending on both PPE concentration and salinity. Herein, sequential ultrafiltration with acetic acid, ethylenediaminetetraacetic acid (EDTA), and water were used for the fabrication of stable nanometer-sized particles. Acetic acid aided removal of metal-ion contamination (palladium and copper) and reduced PPE aggregation in the CPNs by generating repulsive forces. Features in the fluorescence spectrum, such as broadened emission, indicated that the particle formation was driven by aggregation of …