title: Evaluation of cellular uptake and gene transfer efficiency of pegylated poly-L-lysine compacted DNA: implications for cancer gene therapy.
abstract: Recent success in phase I/II clinical trials (Konstan, M. W.; Davis, P. B.; Wagener, J. S.; Hilliard, K. A.; Stern, R. C.; Milgram, L. J.; Kowalczyk, T. H.; Hyatt, S. L.; Fink, T. L.; Gedeon, C. R.; Oette, S. M.; Payne, J. M.; Muhammad, O.; Ziady, A. G.; Moen, R. C.; Cooper, M. J. Hum. Gene Ther. 2004, 15 (12), 1255-69) has highlighted pegylated poly-L-lysine (C1K30-PEG) as a nonviral gene delivery agent capable of achieving clinically significant gene transfer levels in vivo. This study investigates the potential of a C1K30-PEG gene delivery system for cancer gene therapy and evaluates its mode of cellular entry with the purpose of developing an optimally formulated prototype for tumor cell transfection. C1K30-PEG complexes have a neutral charge and form rod-like and toroid-like nanoparticles. Comparison of the transfection efficiency achieved by C1K30-PEG with other cationic lipid and polymeric vectors demonstrates that C1K30-PEG transfects cells more efficiently than unpegylated poly-L-lysine and compares well to commercially available vectors. In vivo gene delivery by C1K30-PEG nanoparticles to a growing subcutaneous murine tumor was also demonstrated. To determine potential barriers to C1K30-PEG gene delivery, the entry mechanism and intracellular fate of rhodamine labeled complexes were investigated. Using cellular markers to delineate the pathway taken by the complexes upon cellular entry, only minor colocalization was observed with EEA-1, a marker of early endosomes. No colocalization was observed between the complexes and the transferrin receptor, which is a marker for clathrin-coated pits. In addition, complexes were not observed to enter late endosomes/lysosomes. Cellular entry of the complexes was completely inhibited by the macropinocytosis inhibitor, amiloride, indicating that the complexes enter cells via macropinosomes. Such mechanistic studies are an essential step to support future rational design of pegylated poly-L-lysine vectors to improve the efficiency of gene delivery.
authors.affiliation: School of Pharmacy, University College Cork, Cork, Ireland. melanie.walsh@ucc.ie
authors.forename: M
authors.initials: M
authors.lastname: Walsh
authors.forename: M
authors.initials: M
authors.lastname: Tangney
authors.forename: M J
authors.initials: MJ
authors.lastname: O'Neill
authors.forename: J O
authors.initials: JO
authors.lastname: Larkin
authors.forename: D M
authors.initials: DM
authors.lastname: Soden
authors.forename: S L
authors.initials: SL
authors.lastname: McKenna
authors.forename: R
authors.initials: R
authors.lastname: Darcy
authors.forename: G C
authors.initials: GC
authors.lastname: O'Sullivan
authors.forename: C M
authors.initials: CM
authors.lastname: O'Driscoll
mesh_terms: D000818:Animals; D019556:COS Cells; D049109:Cell Proliferation; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry / Q000493:pharmacokinetics; D004526:Efficiency; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D053758:Nanoparticles / Q000737:chemistry; D009374:Neoplasms, Experimental / Q000628:therapy; D010873:Pinocytosis; D011092:Polyethylene Glycols / Q000737:chemistry / Q000493:pharmacokinetics*; D011107:Polylysine / Q000737:chemistry / Q000493:pharmacokinetics*; D014162:Transfection; D014184:Transplantation, Homologous
chemical_list: D011107:Polylysine; D011092:Polyethylene Glycols; D004247:DNA