Systemic Delivery of Folate-PEG siRNA Lipopolyplexes with Enhanced Intracellular Stability for in Vivo Gene Silencing in Leukemia

Dian Jang Lee, Eva Kessel, Taavi Lehto, Xueying Liu, Naoto Yoshinaga, Kärt Padari, Ying Chen Chen, Susanne Kempter, Satoshi Uchida, Joachim O. Rädler, Margus Pooga, Ming Thau Sheu, Kazunori Kataoka, Ernst Wagner

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28 引文 斯高帕斯(Scopus)

摘要

Protection of small interfering RNA (siRNA) against degradation and targeted delivery across the plasma and endosomal membranes to the final site of RNA interference (RNAi) are major aims for the development of siRNA therapeutics. Targeting for folate receptor (FR)-expressing tumors, we optimized siRNA polyplexes by coformulating a folate-PEG-oligoaminoamide (for surface shielding and targeting) with one of three lipo-oligoaminoamides (optionally tyrosine-modified, for optimizing stability and size) to generate ∼100 nm targeted lipopolyplexes (TLPs), which self-stabilize by cysteine disulfide cross-links. To better understand parameters for improved tumor-directed gene silencing, we analyzed intracellular distribution and siRNA release kinetics. FR-mediated endocytosis and endosomal escape of TLPs was confirmed by immuno-TEM. We monitored colocalization of TLPs with endosomes and lysosomes, and onset of siRNA release by time-lapse confocal microscopy; analyzed intracellular stability by FRET using double-labeled siRNA; and correlated results with knockdown of eGFPLuc protein and EG5 mRNA expression. The most potent formulation, TLP1, containing lipopolyplex-stabilizing tyrosine trimers, was found to unpack siRNA in sustained manner with up to 5-fold higher intracellular siRNA stability after 4 h compared to other TLPs. Unexpectedly, data indicated that intracellular siRNA stability instead of an early endosomal exit dominate as a deciding factor for silencing efficiency of TLPs. After i.v. administration in a subcutaneous leukemia mouse model, TLP1 exhibited ligand-dependent tumoral siRNA retention, resulting in 65% EG5 gene silencing at mRNA level without detectable adverse effects. In sum, tyrosine-modified TLP1 conveys superior protection of siRNA for an effective tumor-targeted delivery and RNAi in vivo.
原文英語
頁(從 - 到)2393-2409
頁數17
期刊Bioconjugate Chemistry
28
發行號9
DOIs
出版狀態已發佈 - 九月 20 2017

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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