Cellular uptake and intracellular trafficking of polymer conjugates or polymer nanoparticles is typically monitored using fluorescence-based techniques such as confocal microscopy. While these methods have provided a wealth of insight into the internalization and trafficking of polymers and polymer nanoparticles, they require fluorescent labeling of the polymer or polymer nanoparticle. Because in biological media fluorescent dyes may degrade, be cleaved from the polymer or particle, or even change uptake and trafficking pathways, there is an interest in fluorescent label-free methods to study the interactions between cells and polymer nanomedicines. This article presents a first proof-of-concept that demonstrates the feasibility of NanoSIMS to monitor the intracellular localization of polymer conjugates. For the experiments reported here, poly( N-(2-hydroxypropyl) methacrylamide)) (PHPMA) was selected as a prototypical polymer-drug conjugate. This PHPMA polymer contained a 19F-label at the α-terminus, which was introduced in order to allow NanoSIMS analysis. Prior to the NanoSIMS experiments, the uptake and intracellular trafficking of the polymer was established using confocal microscopy and flow cytometry. These experiments not only provided detailed insight into the kinetics of these processes but were also important to select time points for the NanoSIMS analysis. For the NanoSIMS experiments, HeLa cells were investigated that had been exposed to the PHPMA polymer for a period of 4 or 15 h, which was known to lead to predominant lysosomal accumulation of the polymer. NanoSIMS analysis of resin-embedded and microtomed samples of the cells revealed a punctuated fluorine signal, which was found to colocalize with the sulfur signal that was attributed to the lysosomal compartments. The localization of the polymer in the endolysosomal compartments was confirmed by TEM analysis on the same cell samples. The results of this study illustrate the potential of NanoSIMS to study the uptake and intracellular trafficking of polymer nanomedicines.

中文翻译：

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聚（N-（2-羟丙基）甲基丙烯酰胺）的细胞吸收和细胞内贩运。

通常使用基于荧光的技术（例如共聚焦显微镜）来监测聚合物结合物或聚合物纳米颗粒的细胞摄取和细胞内运输。尽管这些方法为聚合物和聚合物纳米颗粒的内在化和运输提供了丰富的见识，但它们需要对聚合物或聚合物纳米颗粒进行荧光标记。因为在生物介质中，荧光染料可能会降解，从聚合物或颗粒上裂解，甚至改变吸收和运输途径，所以人们对无荧光标记的方法感兴趣，以研究细胞与聚合物纳米药物之间的相互作用。本文介绍了第一个概念验证，它证明了NanoSIMS监视聚合物结合物在细胞内定位的可行性。对于此处报告的实验，选择聚（N-（2-羟丙基）甲基丙烯酰胺）（PHPMA）作为原型聚合物-药物偶联物。这种PHPMA聚合物在α端含有19F标记，为了允许进行NanoSIMS分析而引入了该标记。在NanoSIMS实验之前，使用共聚焦显微镜和流式细胞术确定了聚合物的摄取和细胞内运输。这些实验不仅提供了对这些过程动力学的详细了解，而且对于选择NanoSIMS分析的时间点也很重要。对于NanoSIMS实验，研究了已暴露于PHPMA聚合物4或15 h的HeLa细胞，已知这会导致聚合物的溶酶体显着积累。对树脂包埋的和切片的细胞样品进行的NanoSIMS分析表明存在被标记的氟信号，被发现与溶酶体区室的硫信号共定位。通过在相同细胞样品上的TEM分析证实了聚合物在溶酶体区室中的定位。这项研究的结果说明了NanoSIMS在研究聚合物纳米药物的摄取和细胞内运输方面的潜力。