185. Structural Advantage of Dendritic poly(L- lysine) in Gene Delivery In Vitro and In Vivo
Authors: Takuro Niidome, Masato Yamagata, Takayuki Iida, Takahito Kawano, Kouhei Shiba, Yoshiki Katayama
Journal: Molecular Therapy (2006)

Background Recently, we reported that mono-dispersed dendritic poly(L- lysine) of the 6th generation (KG6) showed high transfection efficiency into several cultivated cells with low cytotoxicity (1). The transfection efficiency was not seriously reduced, even when 50% serum was added to the transfection medium. The zeta potential of DNA complex with KG6 was kept at neutral even when the charge ratio (+/−) was increased to 8, supporting the stability in the presence of serum at high concentration. In addition, the complex could circulate in blood flow for long time at least 3h after intravenous injection into mice (2). On the other hand, linear poly(L-lysine) (PLL) doesn't show such high transfection efficiency in vitro and in vivo. In this study, we focused on the structural difference between dendritic poly(L-lysine) and linear poly(L-lysine), and tried to understand the relationship between the structure of poly(L-lysine)s, the physicochemical properties of the DNA complex, and efficiencies of gene delivery. Such a basic study on relationship between structure and function of the gene carrier will provide important clues to find new concept of gene carrier in the next generation. Results and Discussion As dendritic poly(L-lysine) has 128 of amines in the molecule, we employed linear poly(L-lysine) purchased from Sigma (P-2658), which has 170 amines on side chains of the lysine residues, as a contrastive molecule. The poly(L-lysine)s were mixed with plasmid DNA at amine/phosphate ratio of 8, then, zeta-potential and size of the DNA complexes were measured using Zetasizer Nano (Malvern). As a result, KG6 formed neutral DNA complex, while PLL-DNA complex had cationic surface. Size of DNA complex of KG6 was larger than that of PLL, indicating that PLL had advantage in forming highly compacted complex compared with KG6. Uptake of the DNA complex into CHO cells was measured using real time PCR. Larger amount of DNA was detected from the cells transfected with PLL than the case of KG6, indicating that uptake of DNA complex with PLL was higher than that with KG6. Efficiency of gene expression from the plasmid DNA encoding luciferase in cells was quantified by measuring the luciferase activity in the cell lysate. The gene expression mediated by KG6 was higher than that by PLL. Taken in the light of the result of DNA uptake into cells, it is suggested that KG6 had advantage in transferring the DNA from endosomal compartment to cytosol. Conclusion The dendritic structure of poly(L-lysine) was advantageous in; 1) forming neutral complex that invests stability in vitro and in vivo transfection, 2) forming weak DNA complex that make dissociate easily to express the gene, 3) transferring the gene from endosomal compartment to cytosol. References 1. Ohsaki, M., Okuda, T., Wada, A., Hirayama, T., Niidome, T., Aoyagi, H. (2002) Bioconjugate Chem., 13, 510–517. 2. Kawano, T., Okuda, T., Aoyagi, H., Niidome, T. (2004) J. Control. Release, 99, 329–337.