Evaluation of Cellular Uptake and Gene Transfer Efficiency of Pegylated Poly-l-lysine Compacted DNA:  Implications for Cancer Gene Therapy

Abstract

Recent success in phase I/II clinical trials (Konstan, M. W.; Davis, P. B.; Wagener, J. S.; Hilliard, K. A.; Stern, R. C.; Milgram, L. J.; Kowalczyk, T. H.; Hyatt, S. L.; Fink, T. L.; Gedeon, C. R.; Oette, S. M.; Payne, J. M.; Muhammad, O.; Ziady, A. G.; Moen, R. C.; Cooper, M. J. Hum. Gene Ther. 2004, 15 (12), 1255-69) has highlighted pegylated poly-L-lysine (C1K30-PEG) as a nonviral gene delivery agent capable of achieving clinically significant gene transfer levels in vivo. This study investigates the potential of a C1K30-PEG gene delivery system for cancer gene therapy and evaluates its mode of cellular entry with the purpose of developing an optimally formulated prototype for tumor cell transfection. C1K30-PEG complexes have a neutral charge and form rod-like and toroid-like nanoparticles. Comparison of the transfection efficiency achieved by C1K30-PEG with other cationic lipid and polymeric vectors demonstrates that C1K30-PEG transfects cells more efficiently than unpegylated poly-L-lysine and compares well to commercially available vectors. In vivo gene delivery by C1K30-PEG nanoparticles to a growing subcutaneous murine tumor was also demonstrated. To determine potential barriers to C1K30-PEG gene delivery, the entry mechanism and intracellular fate of rhodamine labeled complexes were investigated. Using cellular markers to delineate the pathway taken by the complexes upon cellular entry, only minor colocalization was observed with EEA-1, a marker of early endosomes. No colocalization was observed between the complexes and the transferrin receptor, which is a marker for clathrin-coated pits. In addition, complexes were not observed to enter late endosomes/lysosomes. Cellular entry of the complexes was completely inhibited by the macropinocytosis inhibitor, amiloride, indicating that the complexes enter cells via macropinosomes. Such mechanistic studies are an essential step to support future rational design of pegylated poly-L-lysine vectors to improve the efficiency of gene delivery.

Systemic delivery of therapeutic small interfering RNA using a pH-triggered amphiphilic poly-l-lysine nanocarrier to suppress prostate cancer growth in mice

“…Among the variety of polymers designed for gene delivery, polycationic monodispersed poly(L-lysine) (PLL) emerged as a promising carrier in early studies ( [Walsh et al, 2006 ], [Yusuke et al, 2007], [Meyer et al, 2009] and [Watanabe et al, 2009]) due to controllable molecular size, shape, and the potential for flexible chemical modification. However, because of relatively low transfection efficiency, PLL-based polyplexes are unlikely to be used in clinical applications .…”

Delivery of siRNA and other macromolecules into skin and cells using a peptide enhancer

“…Collectively, these results suggest that macropinocytosis plays a major role in the internalization of SPACE peptide, a conclusion that is shared by other cellpenetrating peptides in the literature (19,20). Studies have reported that cargoes that are internalized by macropinocytosis are often not colocalized with endo/lysosomes implying that their entry into degrading lysosomal compartment can be potentially avoided (21,22). MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, assays on keratinocyte cultures revealed Fig.…”

Stabilization of Plasmid DNA and Lipid-Based Therapeutics as Dehydrated Formulations

“…Dehydration (e.g., lyophilization) has a remarkable influence on the B-(most commonly found DNA conformation under normal physiological conditions) to A-DNA (found under lowhydration conditions) conformational transition [151,152]. Typically, A-and B-DNA vary in the slope of base pairs to the helical axis, major-and minor-groove proportions, crystal packing, and deoxyribose ring conformation [153][154][155]. Studies have reported that during lyophilization, DNA loses its native helical structure [91].…”