International Journal of Biological Macromolecules 50 (2012) 965–973 Contents lists available at SciVerse ScienceDirect International Journal of Biological Macromolecules jo u r n al hom epa ge: ww w.elsevier.com/locate/ijbiomac Glycopolymer modification on physicochemical and biological properties of poly(l-lysine) for gene delivery Dezhong Zhou a , Congxin Li a , Yuling Hu a , Hao Zhou b , Jiatong Chen b , Zhengpu Zhang a , Tianying Guo a,∗ a Key Laboratory of Functional Polymer Materials (Nankai University), Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Tianjin, China b Department of Biochemistry and Molecular Biology, College of Life Science, Nankai University, Weijin Road, No. 94, Tianjin 300071, China a r t i c l e i n f o Article history: Received 23 December 2011 Received in revised form 14 February 2012 Accepted 18 February 2012 Available online 25 February 2012 Keywords: Glycopolymer Poly(l-lysine) Steric effects RAFT polymerization Gene delivery a b s t r a c t Poly(l-lysine) (PLL) has excellent plasmid DNA (pDNA) condensation capacity. However, the relatively high cytotoxicity and low transfection efficiency limit its application as gene delivery vectors. Here, well- defined glycopolymers are synthesized by reversible addition fragmentation transfer polymerization and grafted onto PLL to improve the gene delivery performance. After glycopolymer modification, PLL shows reduced cytotoxicity. By regulating the glycopolymer length and amino group substitution degree, the glycopolymer modified PLL can condense pDNA with proper strength, protect the condensed pDNA from degradation and release them in time. Transfection with NIH3T3 and HepG2 cells shows that the glycopolymer modified PLL has improved transfection efficiencies. The low cytotoxicity, effective pDNA protection and enhanced transfection efficiencies indicate that glycopolymer modification would be an effective strategy to improve the polycation properties for gene delivery. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Over the past two decades, gene therapy has attracted much attention [1]. However, how to design safe and efficient gene deliv- ery vectors is one of the bottlenecks for successful gene therapy [2]. An ideal vector should be (1) safe with low cytotoxicity, (2) of high gene transfer efficiency and (3) specific to the target tis- sues [3]. Gene delivery vectors are divided into viral and non-viral carries. Based on the safety consideration, non-viral vectors have been greatly developed. Natural polycations, such as chitosan, have drawn much attention [4]. As a cationic peptide, poly(l-lysine) (PLL) is favor of interacting with plasmid DNA (pDNA) due to the high positive charge density. However, the application of PLL as gene delivery vector is limited because of the high cytotoxicities [5,6] and low gene transfection efficiencies [7,8]. In order to improve the gene delivery efficiency of the PLL, many attempts have been made. For example, introducing polyethylene glycol (PEG) to reduce the cytotoxicity [9], grafting histidine derivatives to improve the poly- cations/pDNA endosomal escape [10] or tethering target ligands to promoting complex cellular uptake [11,12]. Nevertheless, com- paring with other polycations, such as chitosan (CS), poly(ethylene imine) (PEI) and poly(amido amine) (PAMAM), the applications of PLL are widely limited [13–15]. ∗ Corresponding author. Tel.: +86 22 235 015 97; fax: +86 22 235 015 97. E-mail address: tyguo@nankai.edu.cn (T. Guo). As a cationic polyelectrolyte, the cytotoxicity and gene transfec- tion efficiencies of PLL are relevant to the polycationic properties in biofluid. Saccharides are abundant natural building blocks used to construct many important biological polymers includ- ing proteins, polysaccharides and glycoproteins [16,17]. Herein, we attempted to regulate the PLL polyelectrolyte properties by grafting with well-defined saccharide-containing polymers (gly- copolymers). Influences of the glycopolymer length, amino group substitution degree (SD) on the cytotoxicity and pDNA conden- sation capacity of the modified PLL, polycations/pDNA complex stability and the corresponding gene transfection efficiencies to NIH3T3 and HepG2 cells are investigated. 2. Experimental 2.1. Materials Poly(l-lysine) (PLL, M w = 150–300 kDa), agarose, trypsin–EDTA, Dulbecco’s modified Eagle medium (DMEM), heparin, deoxyri- bonuclease I (DNase), ethidium bromide (EB), fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumerma bromide (MTT) and phosphate buffer solution (PBS, 0.01 M, pH = 7.2–7.4) were obtained from Beijing Dingguo Biotech. Co. Ltd. (Tianjin, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodi- imide hydrochloride (EDC·HCl, 98.5%) and N-hydroxysuccinimide (NHS, analytical grade) were purchased from Aladdin (Shanghai, China). 2,2 ′ -Azobisisobutyronitrile (AIBN) got from Sigma–Aldrich (Shanghai, China) was recrystallized prior to use. Plasmid 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2012.02.021