title: Post-PEGylation of siRNA Lipo-oligoamino Amide Polyplexes Using Tetra-glutamylated Folic Acid as Ligand for Receptor-Targeted Delivery.
abstract: For efficient and receptor-specific siRNA delivery, a new post-PEGylation strategy was established to provide siRNA polyplexes with targeting and shielding agents. For this purpose, core nanoparticles were formed by complexing siRNA with sequence-defined cationic lipo-oligomers. The T-shaped bis-oleoyl-oligoethanamino amides 454 and 595, containing stabilizing tyrosine and cysteine residues, were applied. These core nanoparticles were surface-shielded by reaction with maleimido-polyethylene glycol (Mal-PEG) reagents, optionally containing the targeting ligand folic acid (FolA). The PEGylation had two unpredicted consequences. First, FolA-PEG surface-modified polyplexes agglomerated due to the hydrophobicity of folic acid, resulting in ligand-independent gene silencing. This problem was solved by the use of tetra-γ-glutamyl folic acid (gE4-FolA) as targeting ligand. Post-PEGylated gE4-FolA siRNA polyplexes displayed sizes of 100-200 nm and mediated receptor specific uptake and effective gene silencing. Second, PEGylation triggered a destabilization of polyplexes, which was uncritical in cell culture but a limiting factor in vivo, as revealed by biodistribution studies in mice. This problem was partially overcome by selecting 595 (containing two CRC stability motifs) for polyplex core formation and an optimized lower degree of gE4-FolA PEGylation reagent. Biodistribution in L1210 tumor bearing mice demonstrated a significantly reduced lung signal and extended persistence of siRNA polyplexes (up to 8 h), with moderate delivery into the tumor. Further polyplex stabilization will be required for effective tumor-targeted delivery.
authors.affiliation: Pharmaceutical Biotechnology, Center for System-Based Drug Research, Ludwig-Maximilians-Universität München , Butenandtstrasse 5-13, 81377 Munich, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Müller
authors.affiliation: Pharmaceutical Biotechnology, Center for System-Based Drug Research, Ludwig-Maximilians-Universität München , Butenandtstrasse 5-13, 81377 Munich, Germany.|Nanosystems Initiative Munich , Schellingstrasse 4, D-80799 Munich, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Kessel
authors.affiliation: Pharmaceutical Biotechnology, Center for System-Based Drug Research, Ludwig-Maximilians-Universität München , Butenandtstrasse 5-13, 81377 Munich, Germany.
authors.forename: Philipp M
authors.initials: PM
authors.lastname: Klein
authors.affiliation: Pharmaceutical Biotechnology, Center for System-Based Drug Research, Ludwig-Maximilians-Universität München , Butenandtstrasse 5-13, 81377 Munich, Germany.
authors.forename: Miriam
authors.initials: M
authors.lastname: Höhn
authors.affiliation: Pharmaceutical Biotechnology, Center for System-Based Drug Research, Ludwig-Maximilians-Universität München , Butenandtstrasse 5-13, 81377 Munich, Germany.|Nanosystems Initiative Munich , Schellingstrasse 4, D-80799 Munich, Germany.
authors.forename: Ernst
authors.initials: E
authors.lastname: Wagner
mesh_terms: D000577:Amides / Q000008:administration & dosage* / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002412:Cations / Q000737:chemistry; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems / Q000379:methods; D005260:Female; D005492:Folic Acid / Q000008:administration & dosage* / Q000737:chemistry* / Q000378:metabolism; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007624:KB Cells; D008024:Ligands; D051379:Mice; D008819:Mice, Nude; D053758:Nanoparticles / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism; D011092:Polyethylene Glycols / Q000008:administration & dosage* / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: D000577:Amides; D002412:Cations; D008024:Ligands; D034741:RNA, Small Interfering; D011092:Polyethylene Glycols; D005492:Folic Acid
keywords: folic acid; polyglutamylation; post-PEGylation; sequence defined oligomers; siRNA; targeting ligand