Addressing the instability of DNA nanostructures in tissue culture.
Addressing the instability of DNA nanostructures in tissue culture.
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DOI：
10.1021/nn503513p
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发表时间：
2014-09-23
期刊：
影响因子：
17.1
通讯作者：
Perrault, Steven D.
中科院分区：
文献类型：
--
作者：
Hahn, Jaeseung;Wickham, Shelley F. J.;Shih, William M.;Perrault, Steven D.
关键词：
DNA纳米技术是一种先进的技术，可以为纳米医学提供诊断，治疗和生物医学研究设备。尽管这种装置经常使用体外组织培养模型开发和证明，但这些条件可能与DNA纳米结构的完整性和功能不相容。本研究的目的是表征通过折纸方法产生的3D DNA纳米结构对体外组织培养环境的敏感性，并确定防止纳米结构完整性损失的解决方案。我们研究了细胞培养基的生理阳离子浓度和用作培养基补充剂的胎牛血清（FBS）中存在的核酸酶是否分别导致变性和消化。由于阳离子耗尽导致的DNA纳米结构变性是设计和时间依赖性的，在37 °C下24小时后，四种测试设计中的一种保持完整。通过添加MgSO 4调节培养基可防止变性。在Mg 2+调节的培养基中，FBS核酸酶对纳米结构的消化没有表现出设计依赖性，并且在24小时内变得显著，并且当培养基补充有大于5%FBS时。我们估计补充有10%FBS的培养基在消化DNA纳米结构中含有大于256 U/L当量的DNase I活性。在75 °C下热灭活和在培养基中包含肌动蛋白分别灭活和抑制核酸酶活性。我们研究了培养基调整对细胞生长、活力和表型的影响。将Mg 2+调节至6 mM似乎对细胞没有不利影响。热灭活被认为是不相容的，在体外组织培养，而列入肌动蛋白的生长和活力没有明显的影响。在两个体外试验中，免疫细胞活化和纳米颗粒内吞作用，我们表明，使用与细胞表型和纳米结构完整性相容的条件是获得可靠的实验数据的关键。因此，我们的研究描述了对研究人员进行DNA纳米结构的体外组织培养研究至关重要的考虑因素，以及一些潜在的解决方案，以确保在实验过程中保持纳米结构的完整性和功能。
DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using in vitro tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced via the origami method to the in vitro tissue culture environment and identify solutions to prevent loss of nanostructure integrity. We examined whether the physiological cation concentrations of cell culture medium and the nucleases present in fetal bovine serum (FBS) used as a medium supplement result in denaturation and digestion, respectively. DNA nanostructure denaturation due to cation depletion was design- and time-dependent, with one of four tested designs remaining intact after 24 h at 37 °C. Adjustment of medium by addition of MgSO4 prevented denaturation. Digestion of nanostructures by FBS nucleases in Mg2+-adjusted medium did not appear design-dependent and became significant within 24 h and when medium was supplemented with greater than 5% FBS. We estimated that medium supplemented with 10% FBS contains greater than 256 U/L equivalent of DNase I activity in digestion of DNA nanostructures. Heat inactivation at 75 °C and inclusion of actin protein in medium inactivated and inhibited nuclease activity, respectively. We examined the impact of medium adjustments on cell growth, viability, and phenotype. Adjustment of Mg2+ to 6 mM did not appear to have a detrimental impact on cells. Heat inactivation was found to be incompatible with in vitro tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two in vitro assays, immune cell activation and nanoparticle endocytosis, we show that using conditions compatible with cell phenotype and nanostructure integrity is critical for obtaining reliable experimental data. Our study thus describes considerations that are vital for researchers undertaking in vitro tissue culture studies with DNA nanostructures and some potential solutions for ensuring that nanostructure integrity and functions are maintained during experiments.
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影响因子：
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