Non-toxic phototriggered gene transfection by PAMAM-porphyrin conjugates Ming-Jium Shieh a,b , Cheng-Liang Peng a , Pei-Jen Lou c , Chieh-Hua Chiu a , Tsiao-Yu Tsai d , Chia-Yen Hsu d , Chen-Yu Yeh d , Ping-Shan Lai d, ⁎ a Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No.1, Section 1, Jen-Ai Road, Taipei 100, Taiwan b Department of Oncology, National Taiwan University Hospital and College of Medicine, No. 7, Chung-Shan South Road, Taipei 100, Taiwan c Department of Otolaryngology, National Taiwan University Hospital and College of Medicine, No. 7, Chung-Shan South Road, Taipei 100, Taiwan d Department of Chemistry, National Chung Hsing University, No. 250, Kuo-Kuang Road, Taichung 402, Taiwan ABSTRACT ARTICLE INFO Article history: Received 7 January 2008 Accepted 31 March 2008 Available online 25 April 2008 Keywords: Polyamidoamine dendrimer Photochemical internalization Cytotoxicity Transfection Development of controllable and non-toxic gene transfection systems is a core issue in gene therapy. Photochemical internalization, an innovative strategy in cytosolic release, provides us with an opportunity to develop a light-inducible gene delivery system. In this study, a novel photochemical internalization (PCI)- mediated gene delivery system was synthesized by surface modification of polyamidoamine (PAMAM) dendrimers via 5,10,15-tri(4-acetamidophenyl)-20-mono(4-carboxyl-phenyl)porphyrin (TAMCPP) conju- gated to the generation 4 PAMAM dendrimer (G4). This water-soluble PAMAM-TAMCPP conjugate was characterized for cell viability, phototoxicity, DNA complexation, and in vitro transfection activity. The results show that TAMCPP conjugation did not increase the cytotoxicity of the PAMAM dendrimer below 20 μM, but significantly induced cell death after suitable irradiation. Under almost non-toxic G4-TAMCPP-mediated PCI treatment, the expression of green fluorescent protein determined by flow cytometry could be markedly enhanced in HeLa cells. Therefore, the G4-TAMCPP conjugate had an inducible and effective gene transfection activity, and showed considerable potential as a bimodal biomaterial for PCI-mediated gene therapy. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. 1. Introduction Gene therapy is a promising strategy to deliver desired gene into target cells for the treatment of genetic deficiencies. Viral vectors have been applied in gene delivery because of their high transfec- tion efficacy. However, their safety concerns, including mutagenesis and immunogenicity, affect their broad application in the clinic [1,2]. Non-viral carriers, such as cationic polymer, have been developed as an alternative delivery strategy with less immuno- genicity and lower cost [3,4]. Among these non-viral vectors, polyamidoamine (PAMAM) dendrimer, a novel and unique synthetic macromolecule with a 3-dimensional highly branched structure, is widely used in gene delivery [5,6]. The capability of PAMAM dendrimers to transfect cells appears to depend on the generation and number of primary amino groups on the surface of the polymer. However, generation-dependent cytotoxicity of PAMAM dendri- mers has been shown in previous studies [7,8]. Thus, the develop- ment of PAMAM dendrimers with low generation, low toxicity, and high transfection efficiency is an important area of research in dendrimer-based gene delivery. Many studies have demonstrated that surface modification by hydrophobic moieties, such as fluorescent dyes and amino acids, affect the oligonucleotide or gene transfection activity of PAMAM dendri- mers [9,10]. Although an enhanced activity can be observed with these systems, the transfection process cannot be controlled by a designed switch. Conjugating an appropriate amount of functionalized hydro- phobic molecules to the PAMAM dendrimer to trigger the transfection process and elevate its transfection activity may provide a powerful tool for gene therapy. Photodynamic therapy (PDT) is a photochemical process for producing localized tissue necrosis, which involves the activation of a photosensitizing drug in the target tissue with light of a specific wavelength matched to an absorption peak of the photosensitiser in the presence of molecular oxygen [11]. It is a recognized therapeutic modality, which has regulatory approval for the treatment of a variety of human pre-malignant and malignant diseases. Photochemical internalization (PCI), a specific branch of PDT, is a novel technology utilized for the site-specific release of macromolecules within cells. The mechanism of PCI is based on the breakdown of the endosomal/ lysosomal membranes by photoactivation of photosensitizers that localize on the membranes of these organelles [12]. The PCI strategy has been utilized to release macromolecules such as toxins, DNA delivered as a complex with cationic polymers or incorporated in adenovirus or adeno-associated virus, dendrimer-doxorubicin con- jugates, peptide nucleic acids, and bleomycin, from endocytic vesicles to the cytosol [13–19]. Furthermore, PCI can reverse the adriamycin- resistance of breast cancer cells [20]. Journal of Controlled Release 129 (2008) 200–206 ⁎ Corresponding author. Tel.: +886 4 22840411x409; fax: +886 4 22367269. E-mail address: f89548003@gmail.com (P.-S. Lai). GENE DELIVERY 0168-3659/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2008.03.024 Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel