Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics.
Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics.
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DOI：
10.1038/srep32301
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发表时间：
2016-09-08
影响因子：
4.6
通讯作者：
Dowdy SF
中科院分区：
文献类型：
--
作者：
Lönn P;Kacsinta AD;Cui XS;Hamil AS;Kaulich M;Gogoi K;Dowdy SF
具有生物活性的大分子多肽和寡核苷酸具有显著的治疗潜力。然而，由于它们的大小，它们没有能力进入细胞质。多肽/蛋白转导结构域(PTDS)又称细胞穿透性多肽(CPPs)，可通过内吞作用促进大分子的摄取。然而，克服内体逃逸进入细胞质的限速步骤仍然是一个主要的挑战。已知疏水氨基酸R基团在病毒逃离内体的过程中起着至关重要的作用。在这里，我们利用一种实时、定量的活细胞分裂-GFP荧光互补表型分析方法来系统地分析和优化一系列合成的内体逃逸结构域(EEDs)。通过将EED与TAT-PTD/CPP spilt-GFP多肽互补实验相结合，我们能够通过细胞内分子互补恢复GFP荧光来定量测定内体逃逸到活细胞的细胞质中。我们发现，在与TAT-PTD-Cargo相距6个聚乙二醇基的固定距离上，含有两个芳香族吲哚环或一个吲哚环和两个芳香族苯基的EDS在没有细胞毒性的情况下显著增强了细胞质递送。EDS解决了在将大分子生物肽、蛋白质和siRNA疗法输送到细胞中时内体逃逸的关键限速步骤。
Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells.
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影响因子：
12.4
作者：
Nakase, I;Niwa, M;Futaki, S
通讯作者：
Futaki, S
影响因子：
46.9
作者：
Cabantous, S;Terwilliger, TC;Waldo, GS
通讯作者：
Waldo, GS
DOI：
10.1016/j.bbrc.2015.10.066
发表时间：
2015-11-27
影响因子：
3.1
作者：
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通讯作者：
Kim, Yong-Sung
影响因子：
10.8
作者：
Takayama, Kentaro;Nakase, Ikuhiko;Futaki, Shiroh
通讯作者：
Futaki, Shiroh
影响因子：
15
作者：
Li, Margie;Tao, Yong;Liu, David R.
通讯作者：
Liu, David R.