Chinese Journal of Chemistry, 2007, 25, 1748—1753 Full Paper * E-mail: guopyan@hotmail.com; Tel.: 0086-027-87196030; Fax: 0086-027-87196030 Received August 28, 2006; revised May 23, 2007; accepted July 15, 2007. Project supported by the National Natural Science Foundation of China (No. 29874028), the Hubei Provincial Natural Science Foundation (No. 2006ABA208) and Important Research Project of the Hubei Provincial Department of Education (No. Z200615001). © 2007 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Synthesis and in vitro Property Study of Polyaspartamides YAN, Guo-Ping* ,a (鄢国平) WANG, Xiao-Yan a (王晓燕) WANG, Xu-Li b (王旭立) HUANG, Shi-Wen c (黄世文) ZHUO, Ren-Xi c (卓仁禧) a School of Material Science and Engineering, Wuhan Institute of Technology, Wuhan, Hubei 430073, China b Department of Pharmaceutics and Pharmaceutical Chemistry, School of Pharmacy, University of Utah, Salt Lake City, UT 84108, USA c Department of Chemistry, Wuhan University, Wuhan, Hubei 430072, China Cationic polyaspartamides including poly-α,β-[N'-(2-aminoethy1)-L-aspartamide] (PAEA), poly-α,β-[N'-(4- aminobutyl)-L-aspartamide] (PABA), poly-α,β-[N'-(6-aminohexyl)-L-aspartamide] (PAHA), poly-α,β-[N'-(5-amino- 3-azapentyl)-L-aspartamide] (PAAPA) and poly-α,β-[N'-(8-amino-3,6-diazaoctyl)-L-aspartamide] (PADAOA) were synthesized from polysuccinimide. Their properties were evaluated by 1 H NMR, IR, GPC, fluorescence measure- ment and in vitro cytotoxicity assays. The molecular weights per primary amine charge group of PAEA(1) (M n ＝ 2229), PAAPA and PADAOA are 212, 279, and 226. Polyaspartamides including PAEA(1), PAAPA, PADAOA and low molecular weight PAHA are markedly less toxic than poly(ethyleneimine) and poly(L-lysine), however, PABA and higher molecular weight PAHA are slightly less toxic than poly(L-lysine). Cell cytotoxicity of PAHA was seen to decrease with increasing molecular weight of PAHA, due to water solubility reduction. The negatively charged plasmid DNA has been found to be completely neutralized and complexed by the cationic polyaspar- tamides at an N/P ratio of 5∶1 to 10∶1, forming self-assembled polyplexes via ionic interactions. These polyas- partamide/DNA complexes possess stable zeta potentials and mean particle diameters of about 180 nm for PAEA (1)/DNA and PAAPA/DNA complexes and 280 nm for PADAOA/DNA complexes. Keywords polyaspartamide, cytotoxicity, plasmid DNA, cationic polymer, gene therapy Introduction Non-viral gene therapy using cationic polymers has recently received increased interest as a potential for the treatment of many genetic and non-genetic disorders. This method offers a solution to the problems of vi- ral-based gene delivery systems, such as immunogenic- ity, toxicity, mutagenicity and potential danger of on- cogenicity. 1 Cationic polymers, including poly(L-lysine) (PLL) 2 , poly(ethyleneimine) (PEI) 3 , poly(amidoamine) dendrimer 4 , poly(2-dimethylaminoethyl methacrylate) (pDMAEMA) 5 , chitosan 6 and polyphosphoramidate 7 , all form polyelectrolyte complexes with DNA and are cur- rently under intensive investigation for the non-viral vectors of gene therapy. Poly(ethyleneimine) has successfully been used in gene delivery and possesses high gene transfection. However, accumulation of PEI and its fragments will cause the damage to cell metabolism. At higher concen- trations, it may also mediate disruption of intact cell membranes. 3 Other cationic polymers with lower cell toxicity can not get the ideal gene transfection in vitro or in vivo. Therefore, the design of successful vectors with increasing transfection efficiency in vivo while reducing toxicity is the challenge to efficacious gene therapy. Polyaspartamides are the water-soluble, biologically well-tolerated synthetic polymers with the protein-like structure. They have been proposed as plasma extenders and drug carriers because they are nontoxic, nonanti- genic and degradable in living systems and can be modified easily by reactions with the side chains. 8 As reported, antiviral drugs and anti-inflammatory agents were covalently linked to poly-α,β-[N'-(2-hydroxy-ethyl)- D,L-aspartamide] (PHEA) forming drug-polymer con- jugates capable of increasing drug stability and bioavailility. Therefore polyaspartamides can be used as good polymer carriers for drug delivery. 9 In this work, cationic polyaspartamides including poly-α,β-[N'-(2-aminoethy1)-L-aspartamide] (PAEA), poly-α,β-[N'-(4-aminobutyl)-L-aspartamide] (PABA), poly-α,β-[N'-(6-amino hexyl)-L-aspartamide] (PAHA), poly- α, β-[ N' -(5-amino-3-azapentyl)- L-aspartamide] (PAAPA) and poly-α,β-[N'-(8-amino-3,6-diazaoctyl)- L-aspartamide] (PADAOA) were prepared by the ring open of polysuccinimide with ethylenediamine, 1,4- butylenediamine, 1,6-hexylenediamine, diethylenetria- mine and triethylenetetramine, respectively (Scheme 1). These polyaspartamides were also characterized by 1 H