International Journal of Pharmaceutics 270 (2004) 315–321 Note An assessment of relative transcriptional availability from nonviral vectors Rieko Tachibana a , Naoko Ide a , Yasuo Shinohara a , Hideyoshi Harashima b , C. Anthony Hunt c , Hiroshi Kiwada a,∗ a Faculty of Pharmaceutical Sciences, The University of Tokushima, Shomachi-1-78-1, Tokushima 770-8505, Japan b Department of Molecular Design of Pharmaceutics, Graduate School of Pharmaceutical Sciences, Hokkaido University, 060-8012 Sapporo, Japan c Department of Biopharmaceutical Sciences, The University of California, San Francisco, CA 94143-0446, USA Received 16 March 2003; received in revised form 16 October 2003; accepted 19 October 2003 Abstract To design better delivery systems that enhance transfection efficiency of nonviral vectors, we need to improve our un- derstanding of the mechanisms governing both the amounts of plasmid delivered to the nucleus and gene expression. What is needed is a measure of transcriptional availability (TA): the average level of gene expression per plasmid delivered to the nucleus over the course of an experiment. We describe a method to measure TA and demonstrate its application. The chloramphenicol acetyltransferase reporter gene was transfected into NIH/3T3 cells using either cationic liposomes (TFL-3; O,O ′ -ditetradecanoyl-N-(-trimethylammonioacetyl) diethanolamine chloride (DC-6-14), dioleoylphosphatidylethanolamine (DOPE) and cholesterol, molar ratio 1/0.75/0.75) or cationic polymer (PEI; polyethylenimine). The time courses of both nuclear delivery of plasmids and reporter gene expression were measured for 4 h thereafter. For the conditions used, time courses of gene expression and plasmid nuclear delivery for the two vectors were different. To understand the origins of those differences, we applied a simple pharmacokinetic model, used the data to estimate the values of the model parameters, and interpret differences in estimated parameter values. The rate constant of delivery of plasmids into the nucleus for the TFL-3 vector was twice that of the PEI vector, whereas rate constant of elimination of plasmids in the nucleus for the PEI vector was four times that for the TFL-3 vector. The gene expression rate constant for the TFL-3 vector was estimated to be seven times larger than that of the PEI vector for the conditions used. The pharmacokinetically determined average exposure of a nucleus to plasmid was about 17 times larger for the TFL-3 vector, relative to the PEI vector. That greater exposure resulted in increased relative gene expression. Overall, the TA from the TFL-3 vector was about 13 times greater than from the PEI vector. The experimental design combined with the adoption of pharmacokinetic concepts and principles provide a method to measure TA along with detailed insights into the mechanisms governing gene delivery and expression. © 2003 Elsevier B.V. All rights reserved. Keywords: Nonviral gene delivery; Nuclear delivery; Kinetic analysis; Cationic liposome; Cationic polymer; Polyethylenimine ∗ Corresponding author. Tel.: +81-88-633-7259; fax: +81-88-633-9506. E-mail address: hkiwada@ph.tokushima-u.ac.jp (H. Kiwada). A desire for safe, efficient and effective gene de- livery technologies motivates nonviral gene delivery research. Nonviral gene delivery is expected to have several advantages over viral gene delivery, including low immunogenicity, low acute toxicity, ease of han- 0378-5173/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2003.10.026