Department of Chemistry, University of Toronto , 80 St. George Street, Toronto, Ontario, Canada , M5S 3H6.
Biomacromolecules. 2014 Mar 10;15(3):715-25. doi: 10.1021/bm401483a. Epub 2014 Feb 7.
We describe the synthesis of a heterotelechelic metal-chelating polymer (Bi-MCP-Dox), a polyacrylamide with a number average degree of polymerization DPn = 50 (PDI = 1.2), with biotin (Bi) and doxorubicin (Dox) as functional chain ends and diethylenetriaminepentaacetic acid (DTPA) pendant groups as the binding sites for metal ions. We compared its behavior in cell-uptake experiments with a similar polymer (Bi-MCP) without Dox. These MCPs were complexed with trastuzumab Fab (tmFab) fragments covalently linked to streptavidin (SAv) to form tmFab-SAv-Bi-MCP-Dox and tmFab-SAv-Bi-MCP via the strong affinity between Bi and SAv. tmFab targets human epidermal growth factor receptor-2 (HER2), which is overexpressed on certain human breast cancer cells. Surface plasmon resonance (SPR) experiments with the extracellular domain (ECD) of HER2 showed that incorporation of the MCPs in these complexes had no significant effect on the association or dissociation rate with the HER2 ECD and the dissociation constants. The tmFab-complexed MCPs were subsequently labeled with (111)In (an Auger electron emitting radionuclide). Auger electrons can cause lethal DNA double strand breaks (DSBs) but only if they are emitted intracellularly and especially, in close proximity to the nucleus. To evaluate the cellular and nuclear uptake of tmFab-SAv-Bi-MCP-Dox, we incubated HER2+ SK-BR-3 human breast cancer cells with the complexes saturated with stable In(3+) and visualized their distribution by confocal fluorescence microscopy, monitoring the fluorescence of Dox. In parallel, we carried out cell fractionation studies on tmFab-SAv-Bi-MCP-Dox and on tmFab-SAv-Bi-MCP labeled with (111)In. Both radiolabeled complexes showed cell internalization and nuclear localization. We conclude that metal-chelating polymers with this composition appear to encourage internalization, nuclear uptake, and chromatin (DNA) binding of trastuzumab fragments modified with streptavidin in human breast cancer cells expressing HER2. Further study is needed to understand the impact of polymer charge on cellular uptake and distribution to intracellular compartments.
我们描述了一种杂臂金属螯合聚合物（Bi-MCP-Dox）的合成，该聚合物是一种数均聚合度 DPn = 50（PDI = 1.2）的聚丙烯酰胺，具有生物素（Bi）和阿霉素（Dox）作为功能链端，二乙烯三胺五乙酸（DTPA）作为结合金属离子的基团。我们将其在细胞摄取实验中的行为与具有相似但不含 Dox 的聚合物（Bi-MCP）进行了比较。这些 MCP 与通过链霉亲和素（SAv）共价连接的曲妥珠单抗 Fab（tmFab）片段络合，形成 tmFab-SAv-Bi-MCP-Dox 和 tmFab-SAv-Bi-MCP，这是由于 Bi 和 SAv 之间的强亲和力。tmFab 靶向人表皮生长因子受体-2（HER2），其在某些人类乳腺癌细胞中过度表达。用 HER2 的细胞外结构域（ECD）进行表面等离子体共振（SPR）实验表明，将 MCP 掺入这些复合物中对与 HER2 ECD 的结合或解离速率以及解离常数没有显著影响。随后，用（111）In（一种俄歇电子发射放射性核素）标记与 tmFab 结合的 MCP。俄歇电子只有在细胞内发射，特别是在靠近核的位置，才能导致致命的 DNA 双链断裂（DSB）。为了评估 tmFab-SAv-Bi-MCP-Dox 的细胞和核摄取，我们用稳定的 In(3+)饱和的复合物孵育 HER2+ SK-BR-3 人乳腺癌细胞，并通过共聚焦荧光显微镜监测 Dox 的荧光来观察其分布。同时，我们对 tmFab-SAv-Bi-MCP-Dox 和用（111）In 标记的 tmFab-SAv-Bi-MCP 进行了细胞分馏研究。这两种放射性标记的复合物均显示出细胞内化和核定位。我们得出结论，这种组成的金属螯合聚合物似乎促进了在表达 HER2 的人类乳腺癌细胞中带有链霉亲和素的曲妥珠单抗片段的内化、核摄取和染色质（DNA）结合。需要进一步研究来了解聚合物电荷对细胞摄取和分布到细胞内隔室的影响。
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