Synthesis and Characterization of New Permanently Charged Poly(amidoammonium) Salts and Evaluation of Their DNA Complexes for Gene Transport Pascal Y. Vuillaume,* ,² Me ´ lanie Brunelle, ² Marie-Rose Van Calsteren, ‡ Sylvette Laurent-Lewandowski, ² Andre ´ Be ´ gin, ‡ Raymond Lewandowski, § Brian G. Talbot, | and Youssef ElAzhary ² Universite ´ de Montre ´ al, Faculte ´ de me ´ decine ve ´ te ´ rinaire and Institut de biotechnologie ve ´ te ´ rinaire et alimentaire, 3200 rue Sicotte, Saint-Hyacinthe, Que ´ bec, Canada J2S 2M2, Agriculture et Agroalimentaire Canada, Centre de recherche et de de ´ veloppement sur les aliments, 3600 boul. Casavant Ouest, Saint-Hyacinthe, Que ´ bec, Canada J2S 8E3, 3100 chemin Duplessis, Fleurimont, Que ´ bec, Canada J1H 5H3, and Universite ´ de Sherbrooke, Faculte ´ des sciences, 2500 boul. de l’Universite ´ , Sherbrooke, Que ´ bec, Canada J1K 2R1 Received January 31, 2005; Revised Manuscript Received March 25, 2005 A new series of linear and permanently charged poly(amidoammonium) salts were synthesized in order to investigate the influence of their ionic and hydrophobic contents on both the cytotoxicity and the transfection mediated by polycation-DNA complexes. The poly(amidoammonium) salts were prepared by chemical modification of a parent poly(amidoamine) containing two tertiary amino groups per structural unit: one incorporated into the main chain and the other fixed at the end of a short bismethylene spacer. The permanent charges were introduced through a quaternization reaction involving iodomethane or 1-iodododecane as an alkylating agent. Under appropriate conditions, the methylation reaction was found to be regioselective, allowing the quaternization of either the side chains or both the side chains and the backbone. Under physiological salt conditions (150 mM NaCl), all of the poly(amidoammonium) salts self-assembled with DNA to form complexes. High proportions of highly quaternized polycation provided better defined morphology to the polycation-DNA complexes. Complexes formed from unquaternized polycation were less cytotoxic than branched poly(ethyleneimine) (25 kDa). At high polycation-DNA weight ratios, the introduction of permanent charges generated a significant increase in the cytotoxicity, but no patent correlation could be established with the amount and the position of the permanent charges. Only complexes formed from polycations with quaternized backbone were able to generate significant gene expression, which was putatively attributed to a better defined toroidal-like morphology together with a higher stability, as suggested by zeta potential measurements. The incorporation of dodecane side chains on highly charged polycations severely amplified the cytotoxicity so that, in return, the transfection level was dramatically affected. Introduction The transport and delivery of DNA to cells is of major biological importance for therapeutic applications such as gene therapy and DNA vaccines. So far, viral vectors are the most efficient carriers in delivering DNA into specific organelles. However, the use of viral vectors, as with recombinant viruses, presents serious limitations due to their potential immunogenicity and oncogenicity when used in vivo. 1 Nevertheless, viral vectors have inspired chemists to prepare alternative synthetic vectors. Among the numerous strategies employed to produce DNA-carrier complexes, electrostatic self-assembly between an oppositely charged polycation and DNA is probably one of the most promising methods. These complexes can transfect mammalian cells in vitro and in vivo. 2 Polycation-based DNA complexes represent a simple way to increase gene delivery into cells. However, the main drawback of these nonviral vectors is their relatively low in vivo gene transfection efficiency when compared with viral vectors. There are several explanations for this observation, such as the inability of the complex to be internalized in the cell, 3 the difficulty to permeablize the endosomal membrane, 4 the problems of migration of the complex within the cytoplasm, and its entry into the nucleus. 5,6 Consequently, there is a significant need to design new carriers in order to enhance delivery of DNA and at the same time to better understand the properties that are crucial for efficient transfection. Considerable efforts have been made to design functional polycations capable of overcoming cellular barriers to trans- fection. Poly(-aminoester)s, 7,8 poly(amino alcoholester)s, 9 poly(L-lysine) (PLL), 10 poly(ethyleneimine) (PEI) 11,12 and its conjugates, 13 chitosan, 14 poly(methacrylate)s, 15 aliphatic * To whom correspondence should be addressed. E-mail: p.vuillaume@ sympatico.ca. Phone: (450) 773-8521. Fax: (450) 773-8461. ² Universite ´ de Montre ´al. ‡ Agriculture et Agroalimentaire Canada. § Fleurimont. | Universite ´ de Sherbrooke. 1769 Biomacromolecules 2005, 6, 1769-1781 10.1021/bm050072o CCC: $30.25 © 2005 American Chemical Society Published on Web 04/19/2005