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首页> 外文期刊>International journal of hyperthermia: The official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group >Luciferase-based protein denaturation assay for quantification of radiofrequency field-induced targeted hyperthermia: Developing an intracellular thermometer
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Luciferase-based protein denaturation assay for quantification of radiofrequency field-induced targeted hyperthermia: Developing an intracellular thermometer

机译：基于荧光素酶的蛋白质变性测定法，用于量化射频场诱导的靶向热疗：开发细胞内温度计

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Background: Several studies have reported targeted hyperthermia at the cellular level using remote activation of nanoparticles by radiofrequency waves. To date, methods to quantify intracellular thermal dose have not been reported. In this report we study the relationship between radio wave exposure and luciferase denaturation with and without intracellular nanoparticles. The findings are used to devise a strategy to quantify targeted thermal dose in a primary human liver cancer cell line. Methods: Water bath or non-invasive external Kanzius RF generator (600W, 13.56MHz) was used for hyperthermia exposures. Luciferase activity was measured using a bioluminescence assay and viability was assessed using Annexin V-FITC and propidium iodide staining. Heat shock proteins were analysed using western blot analysis. Results: Duration-dependent luciferase denaturation was observed in SNU449 cells exposed to RF field that preceded measurable loss in viability. Loss of luciferase activity was higher in cetuximab-conjugated gold nanoparticle (C225-AuNP) treated cells. Using a standard curve from water bath experiments, the intracellular thermal dose was calculated. Cells treated with C225-AuNP accumulated 6.07 times higher intracellular thermal dose than the untreated controls over initial 4min of RF exposure. Conclusion: Cancer cells when exposed to an external RF field exhibit dose-dependent protein denaturation. Luciferase denaturation assay can be used to quantify thermal dose delivered after RF exposures to cancer cells with and without nanoparticles.
机译：背景：几项研究报告了通过射频波远程激活纳米粒子，在细胞水平上靶向热疗。迄今为止，尚未报道量化细胞内热剂量的方法。在本报告中，我们研究了有无细胞内纳米颗粒时无线电波暴露与荧光素酶变性之间的关系。这些发现被用于设计一种策略，以量化原发性人类肝癌细胞系中的目标热剂量。方法：使用水浴或非侵入性外部Kanzius RF发生器（600W，13.56MHz）进行热疗暴露。使用生物发光测定法测量荧光素酶活性，并使用膜联蛋白V-FITC和碘化丙啶染色评估生存力。使用蛋白质印迹分析法分析热激蛋白。结果：在暴露于RF场的SNU449细胞中观察到了持续时间依赖性的荧光素酶变性，该过程在可测量的活力丧失之前。在西妥昔单抗偶联的金纳米颗粒（C225-AuNP）处理的细胞中，萤光素酶活性的损失较高。使用水浴实验的标准曲线，计算细胞内热剂量。在最初的RF暴露4分钟内，用C225-AuNP处理的细胞积累的细胞内热剂量比未处理的对照高6.07倍。结论：癌细胞暴露于外部RF场时表现出剂量依赖性蛋白质变性。荧光素酶变性测定可用于量化RF暴露于有或没有纳米颗粒的癌细胞后传递的热剂量。

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