In£uence of hydrophilicity of cationic polymers on the biophysical properties of polyelectrolyte complexes formed by self-assembly with DNA Kenneth A. Howard a , Philip R. Dash a , Martin L. Read a , Kim Ward a , Lesley M. Tomkins b , Ola Nazarova c , Karel Ulbrich c , Leonard W. Seymour a ; * a CRC Institute of Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK b School of Biological Sciences and Biochemistry, University of Birmingham, Birmingham B15 2TT, UK c Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, 2, Heyrovsky Square, Prague 6, 16206 Czech Republic Received 22 December 1999; received in revised form 13 April 2000; accepted 26 April 2000 Abstract To investigate the possibility of producing charge-neutral gene delivery complexes with extended, non-particulate structures, DNA was allowed to self-assemble with a series of hydrophilic cationic polymers containing quaternary charged trimethylammonio ethylmethacrylate (TMAEM, 5, 15, 50, 100 mol%) copolymerised with hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA, 95, 85, 50, 0 mol%, respectively). Copolymers were all able to bind DNA, assessed using ethidium bromide fluorescence, although copolymers with low TMAEM content did not expel ethidium bromide. Increasing TMAEM content of the copolymers changed the morphology of the complexes from extended (5^15 mol% TMAEM), through partially condensed particles (50 mol%) to discrete nanoparticles (100 mol% TMAEM). Complexes based on copolymers with low TMAEM content (5^50 mol%) showed less resistance to degradation by nucleases and lower surface charge (21.2 þ 5.9^45.1 þ 3.9 mV) than those formed using 100 mol% TMAEM (57.8 þ 8.2 mV). They also showed significantly less association with phagocytic cells in vitro (human leucocytes, uptake decreased by up to 92.3% ; murine peritoneal macrophages, uptake decreased by up to 69.6%), although in vivo their hepatic accumulation was only slightly decreased (maximum decrease 27.6%). Finding the appropriate balance of hydrophilicity and stability is key to development of effective vectors for gene delivery. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : DNA; Gene therapy; Gene delivery ; Polyelectrolyte complex ; Phagocytosis ; Poly[2-(trimethylammonio)ethyl methacrylate chloride] ; N-(2-hydroxypropyl)methacrylamide ; Reticulo endothelial system 1. Introduction Polyelectrolyte complexes, formed by self-assembly of cationic polymers with plasmid DNA, are being widely developed as vectors for gene delivery. They show consid- erable versatility, including the possibility for surface- modi¢cation with hydrophilic polymers [1] or linkage of speci¢c targeting ligands for gene expression mediated through speci¢c receptors [2^4]. Factors governing the self-assembly reaction are also well-documented, with charge neutralisation leading to a fall in hydrophilicity as the polyelectrolyte components self-assemble. Depend- ing on the properties of the cationic polymer backbone, charge-neutralised regions of the complex often become hydrophobic and tend to aggregate in aqueous solvents, driving spontaneous formation of insoluble particles [5]. The net result is that simple polyelectrolyte complexes formed in water between low concentrations of DNA and most polycations are usually discrete particles with size ranging from 25^200 nm diameter [6]. DNA within polyelectrolyte nanoparticles is usually protected from degradation by serum nucleases, although it is capable of undergoing e¤cient transcription within the nucleus [7]. These properties are useful for transfection in vitro, although di¤cult to exploit in vivo because the complexes are avidly phagocytosed by cells of the reticulo- endothelial system (RES). Following intravenous adminis- tration, for example, poly(L-lysine) (pLL)/DNA complexes are rapidly sequestered by the Kup¡er cells, with over 70% 0304-4165 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII:S0304-4165(00)00076-3 * Corresponding author. Fax: +44-121-414-3263; E-mail : l.seymour@bham.ac.uk Biochimica et Biophysica Acta 1475 (2000) 245^255 www.elsevier.com/locate/bba