title: Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/SiRNA polyplexes.
abstract: Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA polyplexes were characterized. Stability and size of siRNA polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA polyplexes.
authors.affiliation: Department of Pharmaceutics and Biopharmacy, Philipps Universität Marburg, Ketzerbach 63, 35032 Marburg, Germany.
authors.forename: Shirui
authors.initials: S
authors.lastname: Mao
authors.forename: Michael
authors.initials: M
authors.lastname: Neu
authors.forename: Oliver
authors.initials: O
authors.lastname: Germershaus
authors.forename: Olivia
authors.initials: O
authors.lastname: Merkel
authors.forename: Johannes
authors.initials: J
authors.lastname: Sitterberg
authors.forename: Udo
authors.initials: U
authors.lastname: Bakowsky
authors.forename: Thomas
authors.initials: T
authors.lastname: Kissel
mesh_terms: D000818:Animals; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D007097:Imines / Q000737:chemistry*; D046911:Macromolecular Substances; D051379:Mice; D018625:Microscopy, Atomic Force; D041681:NIH 3T3 Cells; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry*; D011095:Polyethylenes / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism
chemical_list: D007097:Imines; D046911:Macromolecular Substances; D011095:Polyethylenes; D034741:RNA, Small Interfering; C505405:poly(ethylene imine); D011092:Polyethylene Glycols; D006493:Heparin