The gene transfection efﬁciency of a folate–PEI600–cyclodextrin nanopolymer Hong Yao a, b , Samuel S. Ng b , Wesley O. Tucker b , Yuk-Kai-Tiu Tsang b , Kwan Man c , Xiao-mei Wang d , Billy K.C. Chow e , Hsiang-Fu Kung a, g , Gu-Ping Tang b, f, ** , Marie C. Lin a, b, * a Biomedical Engineering Research Centre, Kunming Medical University, Kunming, PR China b Integrative Chemical Biology Laboratory, Department of Chemistry, The University of Hong Kong, Hong Kong c Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong d Medical College, Shenzhen University, Shenzhen, PR China e School of Biological Sciences, The University of Hong Kong, Hong Kong f Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou, PR China g Stanley Ho Center for Emerging Infectious Diseases, The State Key Laboratory in Oncology in South China, The Chinese University of Hong Kong, Hong Kong article info Article history: Received 30 April 2009 Accepted 26 June 2009 Available online 16 July 2009 Keywords: Polyethylenimine b-Cyclodextrin Folate Gene therapy Biodegradation In vivo test abstract The success of gene therapy relies on a safe and effective gene delivery system. In this communication, we describe the use of folate grafted PEI 600 –CyD (H 1 ) as an effective polyplex-forming plasmid delivery agent with low toxicity. The structures of the polymer and polyplex were characterized, and the in vitro transfection efﬁciency, cytotoxicity, and in vivo transfection of H 1 were examined. We found that folate molecules were successfully grafted to PEI 600 –CyD. At N/P ratios between 5 and 30, the resulting H 1 /DNA polyplexes had diameters less than 120 nm and zeta potentials less than 10 mV. In various tumor cell lines examined (U138, U87, B16, and Lovo), the in vitro transfection efﬁciency of H 1 was more than 50%, which could be improved by the presence of fetal bovine serum or albumin. The cytotoxicity of H 1 was signiﬁcantly less than high molecular weight PEI-25 kDa. Importantly, in vivo optical imaging showed that the efﬁciency of H 1 -mediated transfection (50 mg luciferase plasmid (pLuc), N/P ratio ¼ 20/1) was comparable to that of adenovirus-mediated luciferase transduction (1  10 9 pfu) in melanoma-bearing mice, and it did not induce any toxicity in the tumor tissue. These results clearly show that H 1 is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA and its application warrants further investigation. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Gene therapy has evolved as a promising therapeutic strategy for cancer and various intractable human diseases. The efﬁcacy and safety of gene therapy depend not only on gene construct being delivered, but more importantly on the delivery vehicle itself. Previously, viral vectors have been a primary focus for cancer gene delivery due to their excellent transfection efﬁciency in vitro and in vivo [1,2]. However, because of their potential safety risks [3,4] and immunogenicity [5], there is an urgent need to develop alternative non-viral gene carriers. Hence, cationic liposomal and polymeric vectors are common non-viral vectors currently being developed. Unlike viral vectors, these non-viral alternatives offer several advantages over viral vectors. These include increased safety, ease of design and synthesis, low production cost, and ﬂexibility in chem- ical modiﬁcations for improved biocompatibility and target-speci- ﬁcity. A number of cationic polymers have been demonstrated to display considerable transfection efﬁciency in vivo and in vitro in the past decade. Among those cationic polymers, polyethylenimine (PEI) has emerged as a promising delivery reagent, primarily owing to its excellent transfection efﬁciency in a wide range of cell types [6]. By virtue of its abundant primary amines, PEI readily forms polyplexes with negatively charged DNA and subsequently buffers the endo- somal environment, facilitating the release of DNA in the cytosol [7,8]. It should be noted that the transfection efﬁciency and cyto- toxicity of PEI are molecular weight-dependent. In general, PEI with higher molecular weight has higher transfection efﬁciency, but also higher cytotoxicity. Furthermore, PEI has a propensity to form aggregates in physiological conditions [7,9]. To address this issue, polyethylene glycol (PEG) grafting to PEI has been found useful in reducing cytotoxicity, although PEGylation did not enhance * Corresponding author. Department of Chemistry, Room 8N11, 8/F Kadoorie Biological Sciences Building, The University of Hong Kong, Pokfulam Road, Hong Kong. Tel.: þ852 2299 0776. ** Corresponding author. Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou 310028, PR China. E-mail addresses: tangguping@yahoo.com.cn (G.-P. Tang), mcllin@hku- sua.hku.hk (M.C. Lin). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.06.051 Biomaterials 30 (2009) 5793–5803