Electrostatically Mediated Interactions between Cationic Lipid–DNA Particles and an Anionic Surface 1 Frances M. P. Wong,* , † ,2 Marcel B. Bally,* , † and Donald E. Brooks* , ‡ *Department of Pathology and Laboratory Medicine and ‡Department of Chemistry, University of British Columbia, 2211 Wesbrook Mall, Vancouver, British Columbia, V6T 2B5, Canada; and †Department of Advanced Therapeutics, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver, British Columbia, V5Z 1L3, Canada Received November 30, 1998, and in revised form March 9, 1999 In an effort to model the interaction of lipid-based DNA delivery systems with anionic surfaces, such as a cell membrane, we have utilized microelectrophoresis to characterize how electrokinetic measurements can provide information on surface charge and binding characteristics. We have established that cationic lip- ids, specifically N-N-dioleoyl-N,N-dimethylammonium chloride (DODAC), incorporated into liposomes pre- pared with 1,2-dioleoyl-i-glycero-3-phosphoethanol- amine (DOPE) or 1,2-dioleoyl-sn-glycero-3-phospho- choline (DOPC) at 50 mol%, change the inherent elec- trophoretic mobility of anionic latex polystyrene beads. Self-assembling lipid–DNA particles (LDPs), prepared at various cationic lipid to negative DNA phosphate charge ratios, effected no changes in bead mobility when the LDP charge ratio (/) was equal to or less than 1. Increasing the LDP concentration in a solution of 0.1% (w/v) anionic beads resulted in a charge reversal effect when a net charge of LDP to total bead charge ratio (/) of 1:1 was observed. LDP formulations, utilizing either DOPE or DOPC, showed similar titration profiles with a charge reversal ob- served at a 1:1 net LDP to bead charge ratio (/). It was confirmed through centrifugation studies that the DNA in the LDP was associated with the anionic latex beads through electrostatic interactions. LDP bind- ing, rather than the binding of dissociated cationic lipids, resulted in the observed electrophoretic mobil- ity changes of the anionic latex beads. © 1999 Academic Press Key Words: lipoplex; lipid-DNA particles; electro- static interactions; microelectrophoresis. Transfection of cells, utilizing lipid-based gene transfer methods, involves a number of well-defined steps (1). Each step must be successfully achieved so that a gene may be delivered to the cell of interest. A critical first step in transfection requires delivery of the gene transfer system, a process that involves attach- ment of the delivery system and the associated gene to the cell membrane. Once the gene has reached the desired cell population, it must enter the cell and tra- verse to the nucleus where it may be efficiently tran- scribed and translated such that gene expression can be observed. For lipid-based delivery systems, cationic lipids have been utilized to achieve efficient association of anionic DNA with the carriers (2, 3). Formulation strategies have mainly involved the use of lipids in the form of liposomes (4, 5). However, these strategies have not reached their full potential due to problems caused by DNA cross-linking of liposomes and liposome fusion (6). These reactions are difficult to control and result in formation of large heterogeneous aggregates. Alterna- tively, we have developed novel self-assembling lipid– DNA particles for gene transfer (7). These formulations are structurally distinct from liposomal-based systems and have the advantages of being small, well-defined, and homogeneous. Regardless of the technology used for gene transfer, efficient transfection must involve successful binding of the delivery vehicle to the cell membrane. Delivery and attachment of cationic lipid gene transfer vehicles have been suggested to be mediated by electrostatic interactions (8). Characterization of the electrostatic interaction between the anionic surface and cationic 1 This work was supported by grants from the Medical Research Council of Canada (M.B.B) and Inex Pharmaceuticals Corp. F.M.P.W. holds a studentship from the Cancer Research Society, Inc., and a GREAT Award from the Science Council of B.C. 2 To whom correspondence should be addressed. Fax: (604) 877- 6011. E-mail: fmpwong@bccancer.bc.ca. 0003-9861/99 $30.00 31 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 366, No. 1, June 1, pp. 31–39, 1999 Article ID abbi.1999.1201, available online at http://www.idealibrary.com on