Evaluating Endosomal Escape of Caspase-3-Containing Nanomaterials Using Split GFP.
Evaluating Endosomal Escape of Caspase-3-Containing Nanomaterials Using Split GFP.
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DOI：
10.1021/acs.biomac.0c01767
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发表时间：
2021-03-08
影响因子：
6.2
通讯作者：
Thayumanavan S
中科院分区：
文献类型：
--
作者：
Anson F;Liu B;Kanjilal P;Wu P;Hardy JA;Thayumanavan S
生物制品获得细胞内靶点的能力取决于活性的、未修饰的蛋白质的移位。这通常是使用纳米级的配方实现的，这些配方通过内吞作用进入细胞。这种摄取机制往往限制了生物制品的治疗潜力，因为纳米载体逃脱内噬体体的倾向成为关键决定因素。因此，为了适当地评估和比较不同成分的竞争递送系统，评估内体逃逸效率是至关重要的。不幸的是，缺乏评估胞体逃逸的定量工具，标准方法经常导致对胞浆定位的错误解释。在这项研究中，我们使用分裂互补内体逃逸试验(SEE)来评估聚合物纳米凝胶和介孔二氧化硅纳米颗粒输送后胞浆caspase-3的水平。特别是，我们使用SEE作为一种手段，以实现对聚合物组成、聚合物结构(随机与嵌段)、疏水性和表面功能性的影响的系统研究。虽然聚合物结构对内体逃逸的影响不大，但阳离子和pH敏感多肽的纳米凝胶功能化显著增加了内体逃逸水平，进而显著增加了每个内体的纳米凝胶量。这项工作为开发最佳的caspase-3递送系统提供了指导，因为这种caspase-3变体可以很容易地在任何导致胞浆积累和货物释放的系统中替代治疗性caspase-3货物。此外，这些数据提供了一个框架，可以很容易地应用于各种蛋白质货物，以评估各种蛋白质输送载体摄取和内体逃逸的独立贡献。
The ability for biologics to access intracellular targets hinges on the translocation of active, unmodified protein. This is often achieved using nanoscale formulations, which enter cells through endocytosis. This uptake mechanism often limits the therapeutic potential of the biologics, as the propensity of the nanocarrier to escape the endosome becomes the key determinant. To appropriately evaluate and compare competing delivery systems of disparate compositions, it is therefore critical to assess endosomal escape efficiencies. Unfortunately, quantitative tools to assess endosomal escape are lacking and standard approaches often lead to erroneous interpretation of cytosolic localization. In this study, we use a split-complementation endosomal escape (SEE) assay to evaluate levels of cytosolic caspase-3 following delivery by polymer nanogels and mesoporous silica nanoparticles. In particular, we use SEE as a means to enable systematic investigation of the effect of polymer composition, polymer architecture (random vs. block), hydrophobicity, and surface functionality. Although polymer structure had little influence on endosomal escape, nanogel functionalization with cationic and pH-sensitive peptides significantly enhanced endosomal escape levels and further, significantly increased the amount of nanogel per endosome. This work serves as a guide for developing an optimal caspase-3 delivery system, as this caspase-3 variant can be easily substituted for a therapeutic caspase-3 cargo in any system that results in cytosolic accumulation and cargo release. In addition, these data provide a framework that can be readily applied to a wide variety of protein cargos to assess the independent contributions of both uptake and endosomal escape of a wide range of protein delivery vehicles.
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影响因子：
5.3
作者：
Galliani, Marianna;Tremolanti, Chiara;Signore, Giovanni
通讯作者：
Signore, Giovanni
影响因子：
6.2
作者：
Jiang, Ziwen;He, Huan;Thayumanavan, S.
通讯作者：
Thayumanavan, S.
影响因子：
10.6
作者：
Forman, Henry Jay;Zhang, Hongqiao;Rinna, Alessandra
通讯作者：
Rinna, Alessandra
影响因子：
15
作者：
Gonzalez-Toro, Daniella C.;Ryu, Ja-Hyoung;Chacko, Reuben T.;Zhuang, Jiaming;Thayumanavan, S.
通讯作者：
Thayumanavan, S.
影响因子：
3.8
作者：
Bale, Shyam Sundhar;Kwon, Seok Joon;Dordick, Jonathan S.
通讯作者：
Dordick, Jonathan S.