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Bioconjug Chem. 2017 Sep 20;28(9):2393-2409. doi: 10.1021/acs.bioconjchem.7b00383. Epub 2017 Aug 24.

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

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Department of Pharmacy and Center for NanoScience, Ludwig-Maximilians-Universität München , Butenandtstr. 5-13, 81377 Munich, Germany.
Nanosystems Initiative Munich (NIM) , Schellingstr. 4, 80799 Munich, Germany.
Innovation Center of NanoMedicine (iCONM), Institute of Industry Promotion-Kawasaki , 3-25-14 Tonomachi, Kawasaki-ku, 210-0821 Kawasaki, Japan.
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, 113-8656 Tokyo, Japan.
Institute of Molecular and Cell Biology and Institute of Technology, University of Tartu , 23 Riia Str., 51010 Tartu, Estonia.
School of Pharmacy, College of Pharmacy, Taipei Medical University , No. 250, Wuxin St., 11031 Taipei, Taiwan.
Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-Universität München , Geschwister-Scholl-Platz 1, 80539 Munich, Germany.
Policy Alternatives Research Institute, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, 113-0033 Tokyo, Japan.


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.

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