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Intracellular DNA Cargo Release from a Gold Nanoparticle Modulated by the Nature of the Surface Coupling Functionality
Bioconjugate Chemistry ( IF 4.7 ) Pub Date : 2018-09-10 00:00:00 , DOI: 10.1021/acs.bioconjchem.8b00575
Kate J. F. Carnevale 1 , Geoffrey F. Strouse 1
Affiliation  

Covalently coupling nucleic acids to a gold nanoparticle (AuNP) surface has been demonstrated for generating effective gene therapy agents to modify cellular protein expression. The therapeutic efficacy of the approach is anticipated to be impacted by the length of time the nucleic acid sequence resides in the endolysosomal pathway once transfected into a cell. It is believed that the dynamics of the processing should reflect the linkage chemistry of the DNA to the AuNP surface. In this manuscript the dynamics of nanotherapeutic uptake, nucleic acid release, and gene processing are investigated in vitro for a AuNP-nucleic acid delivery platform transfected into A375 human melanoma cells, as a function of the nucleic acid-gold linkage chemistry. The dynamics of cell processing of the single monodentate thiol (SX), bidentate dual thiol (SS), or mixed bidentate thiol plus amine (SN) coordination of nucleic acids to the AuNP surface are evaluated using a multicolor nanosurface energy transfer bio-optical transponder (SET-BOT) technology. The use of live-cell fluorescence microscopy allows for the direct visualization of the uptake and localization of a lipofectamine-packaged SET-BOT using a dye (DL700) that is not quenched in the proximity of the AuNP, while fluorescence “turn on” of a dye that is proximally quenched by the AuNP (DL488) is used to report on the dynamics of release of the nucleic acid cargo within the cell. For protein expression following transcription of the gene, the emission signature of a red fluorescent protein, tdTomato, is monitored. The intracellular rates of DNA release from the AuNP surface once endosomally packaged within the A375 human melanoma cells were found to follow the binding activity series: bidentate thiol > bidentate thiol plus amine > monodentate thiol, consistent with the strength of multidentate chelation, paired with the stabilizing influence of π-backbonding of thiols compared to σ-donation in amines, when bound to a gold surface.

中文翻译:

从表面耦合功能的性质调节金纳米粒子的细胞内DNA释放。

已经证明将核酸与金纳米粒子(AuNP)表面共价偶联可产生有效的基因治疗剂来修饰细胞蛋白表达。预期该方法的治疗功效将受核酸序列一旦转染入细胞后停留在溶酶体途径中的时间长度的影响。据信,加工的动力学应反映DNA与AuNP表面的键合化学。在该手稿纳米治疗摄取,核酸释放和基因处理的动力学进行了研究 在体外转染到A375人黑素瘤细胞中的AuNP核酸递送平台的功能,取决于核酸-金连接化学的功能。使用多色纳米表面能量转移生物光学应答器评估核酸与AuNP表面的单单硫醇(SX),双齿双硫醇(SS)或双齿双硫醇加胺(SN)配位的细胞动力学(SET-BOT)技术。活细胞荧光显微镜的使用允许直接染色,使用在AuNP附近未淬灭的染料(DL700)脂质体包装的SET-BOT脂质体的摄取和定位,而荧光的“开启”被AuNP(DL488)近端淬灭的染料用于报告核酸在细胞内释放的动力学。对于基因转录后的蛋白质表达,监测红色荧光蛋白tdTomato的发射特征。内吞包装在A375人黑素瘤细胞中后,发现从AuNP表面释放的DNA的细胞内速率遵循结合活性系列:双齿硫醇>齿双硫醇加胺>单齿硫醇,与多齿螯合的强度一致,与当与金表面结合时,与胺中的σ-给体相比,硫醇的π-回键的稳定影响。
更新日期:2018-09-10
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