title: Freeze-dried formulations for in vivo gene delivery of PEGylated polyplex micelles with disulfide crosslinked cores to the liver.
abstract: A stable, freeze-dried formulation consisting of a core-shell-type polyplex with a poly(ethylene glycol) (PEG) shell (polyplex micelles) was prepared from a polyion complex of plasmid DNA (pDNA) and thiolated PEG-poly(L-lysine) block copolymers. The use of lyoprotectants was avoided by crosslinking the core with disulfide bonds. The crosslinked polyplex micelles (CPMs) showed excellent stability during freeze-drying and reconstitution processes, which is in sharp contrast with the formation of visible agglomerates from the non-crosslinked polyplex micelles (NCPMs) after a similar process. A thiolation degree higher than 13% of the lysine residues was required to achieve sufficient tolerability of the CPMs during the freeze-drying/reconstitution cycle. Dynamic light scattering measurements and atomic force microscopy observations demonstrated that the original size and shape of the CPMs with a thiolation degree of higher than 13% were maintained even after the freeze-drying. Furthermore, the CPMs reconstituted from the freeze-dried state achieved a transfection efficiency as high as that of the original samples. The intravenous injection of the CPM with a thiolation degree of 37% into mice via the orbital vein led to an appreciably uniform gene expression of a yellow fluorescence protein variant (Venus) in the liver, while no gene expression was observed in the case of the free pDNA injection. The procedure of disulfide crosslinking of the polyplex micell core allows the preparation of non-viral gene vectors as a powder formulation without the use of any lyoprotectants. This achievement is certainly useful for pharmaceutical applications and exhibits many advantages, including easy concentration adjustments of dosing samples, long-term storage stability, and large-scale production reproducibility.
authors.affiliation: Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Japan.
authors.forename: Kanjiro
authors.initials: K
authors.lastname: Miyata
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kakizawa
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Nishiyama
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Yamasaki
authors.forename: Tsunamasa
authors.initials: T
authors.lastname: Watanabe
authors.forename: Michinori
authors.initials: M
authors.lastname: Kohara
authors.forename: Kazunori
authors.initials: K
authors.lastname: Kataoka
mesh_terms: D000818:Animals; D002460:Cell Line; D002626:Chemistry, Pharmaceutical; D003432:Cross-Linking Reagents; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004354:Drug Screening Assays, Antitumor; D005079:Excipients; D005612:Freeze Drying; D018014:Gene Transfer Techniques; D006801:Humans; D008027:Light; D008099:Liver / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D008823:Micelles; D018625:Microscopy, Atomic Force; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry*; D011107:Polylysine / Q000031:analogs & derivatives / Q000737:chemistry; D012542:Scattering, Radiation; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D014162:Transfection
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D005079:Excipients; D008823:Micelles; C117924:mPEG-block-PLL; D011107:Polylysine; D011092:Polyethylene Glycols; D004247:DNA; D008156:Luciferases