Using Fluorescence Lifetime Imaging Microscopy to Monitor Theranostic Nanoparticle Uptake and Intracellular Doxorubicin Release
Using Fluorescence Lifetime Imaging Microscopy to Monitor Theranostic Nanoparticle Uptake and Intracellular Doxorubicin Release
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DOI：
10.1021/nn404407g
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发表时间：
2013-11-01
期刊：
影响因子：
17.1
通讯作者：
Davis, Thomas P.
中科院分区：
文献类型：
--
作者：
Basuki, Johan S.;Duong, Hien T. T.;Davis, Thomas P.
我们描述了氧化铁纳米粒子（IONP）的合成，具有优异的胶体稳定性，在水和血清中，赋予精心设计的接枝聚合物壳。聚合物壳构建有连接的醛官能团，以使得能够通过亚胺键可逆地连接多柔比星（DOX），从而在酸性环境中提供DOX的受控释放机制。显示IONP容易被细胞系（MCF-7乳腺癌细胞和H1299肺癌细胞）吸收，并且使用体外荧光寿命成像显微镜（FLIM）测量来证明DOX的细胞内释放。使用天然DOX（类似于1 ns）与缀合DOX（类似于4.6ns）相比表现出的荧光寿命差异，在H1299中原位监测缀合DOX的细胞内释放，并使用相量图表示法进行估计，显示天然DOX随时间的明显增加。使用共聚焦显微镜证实了从FLIM获得的结果，清楚地显示了DOX在细胞核中的积累。IONP也被评估为MRI阴性造影剂。我们观察到IONP的横向弛豫率特性发生了显着变化，在存在或不存在结合DOX的情况下，从220到390 mM（-1）s（-1）。MRI信号对IONP-DOX/水相互作用的这种依赖性可以在未来的治疗诊断应用中被利用。然后将体外研究扩展到使用多光子激发显微镜监测DOX负载的IONP（IONP@P（HBA）-b-P（OEGA）+ DOX）进入从两个独立细胞系（MCF-7和H1299）生长的两个3D多细胞肿瘤球状体（MCS）的细胞摄取。
We describe the synthesis of iron oxide nanoparticles (IONPs) with excellent colloidal stability in both water and serum, imparted by carefully designed grafted polymer shells. The polymer shells were built with attached aldehyde functionality to enable the reversible attachment of doxorubicin (DOX) via imine bonds, providing a controlled release mechanism for DOX in acidic environments. The IONPs were shown to be readily taken up by cell lines (MCF-7 breast cancer cells and H1299 lung cancer cells), and intracellular release of DOX was proven using in vitro fluorescence lifetime imaging microscopy (FLIM) measurements. Using the fluorescence lifetime difference exhibited by native DOX (similar to 1 ns) compared to conjugated DOX (similar to 4.6 ns), the intracellular release of conjugated DOX was in situ monitored in H1299 and was estimated using phasor plot representation, showing a clear increase of native DOX with time. The results obtained from FLIM were corroborated using confocal microscopy, clearly showing DOX accumulation in the nuclei. The IONPs were also assessed as MRI negative contrast agents. We observed a significant change in the transverse relaxivity properties of the IONPs, going from 220 to 390 mM(-1) s(-1) in the presence or absence of conjugated DOX. This dependence of MRI signal on IONP-DOX/water interactions may be exploited in future theranostic applications. The in vitro studies were then extended to monitor cell uptake of the DOX loaded IONPs (IONP@P(HBA)-b-P(OEGA) + DOX) into two 3D multicellular tumor spheroids (MCS) grown from two independent cell lines (MCF-7 and H1299) using multiphoton excitation microscopy.