Tumoral gene silencing by receptor-targeted combinatorial siRNA polyplexes.
Small interfering RNA (siRNA) promises high efficacy and excellent specificity to silence the target gene expression, which shows potential for cancer treatment. However, systemic delivery of siRNA with selectivity to the tumor site and into the cytosol of tumor cells remains a major limitation. To achieve this, we generated oligoaminoamide-based sequence-defined polycationic oligomers by solid-phase assisted synthesis, which can form polyplexes with anionic siRNA by electrostatic interaction to serve as siRNA carrier. Targeting for folate receptor (FR)-overexpressing tumors, we optimized the physicochemical properties of polyplexes by combinatorial optimization of PEGylated folate-conjugated oligomer (for FR targeting and shielding of surface charges) and 3-arm oligomer (for size modification and particle stability). For uni-directional fast coupling between the two groups of oligomers, we activated the cysteine thiol groups of one of the oligomers with 5,5'-dithio-bis(2-nitrobenzoic acid) to achieve a fast chemical linkage through disulfide formation with the free thiol groups of the other oligomer. These targeted combinatorial polyplexes (TCPs) are homogeneous spherical particles with favorable size and surface charge, which showed strong siRNA binding activity. TCPs were internalized into cells by FR-mediated endocytosis, triggered significant eGFP-luciferase marker gene silencing, and transfection with antitumoral EG5 siRNA suppressed cell proliferation in FR-expressing tumor cells. Moreover, the most promising formulation TCP1 after i.v. administration in tumor-bearing mice exhibited siRNA delivery into the tumor, resulting in EG5 gene silencing at mRNA level. Therefore, by covalent combination of two sequence-defined functional oligomers, we developed a siRNA carrier system with optimized size and surface charge for efficient tumor cell-directed gene silencing and cytotoxicity in vitro and in vivo.
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.
Dian-Jang
DJ
Lee
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.
Dongsheng
D
He
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.
Eva
E
Kessel
Institute of Molecular and Cell Biology, University of Tartu, 23 Riia Str., 51010 Tartu, Estonia.
Kärt
K
Padari
Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-Universität München, Geschwister-Scholl-Platz 1, 80539 Munich, Germany.
Susanne
S
Kempter
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.
Ulrich
U
Lächelt
Nanosystems Initiative Munich (NIM), Schellingstr. 4, 80799 Munich, Germany; Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-Universität München, Geschwister-Scholl-Platz 1, 80539 Munich, Germany.
Joachim O
JO
Rädler
Institute of Molecular and Cell Biology, University of Tartu, 23 Riia Str., 51010 Tartu, Estonia.
Margus
M
Pooga
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. Electronic address: ernst.wagner@cup.uni-muenchen.de.
Ernst
E
Wagner
D000818:Animals; D045744:Cell Line, Tumor; D005260:Female; D058972:Folic Acid Transporters / Q000378:metabolism*; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016547:Kinesins / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D008819:Mice, Nude; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism*; D011108:Polymers / Q000008:administration & dosage / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000008:administration & dosage / Q000737:chemistry
D058972:Folic Acid Transporters; C470243:KIF11 protein, human; D011108:Polymers; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013438:Sulfhydryl Compounds; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases; D016547:Kinesins
Endosomal escape; Folate receptor; Polyplexes; Tumor targeting; siRNA