Impact of Cationic Charge Density and PEGylated Poly(Amino Acid) Tercopolymer Architecture on Their Use as Gene Delivery Vehicles. Part 2: DNA Protection, Stability, Cytotoxicity, and Transfection Efficiency.

The impact of the molecular architecture on the transfection efficiency of PEGylated poly(amino acid) block copolymers was investigated for PEG-b-p(L-Lys)(x)-b-p(L-Leu)(y), PEG-b-p(L-Leu)(x)-b-p(L-Lys)y, and PEG-b-p((L-Leu)(x)-co(L-Lys)(y)). The block lengths of p(L-Lys) and p(L-Leu) were varied between 10, 20, and 40; and 10 and 20, respectively, to study the influence of the ionic/hydrophobic balance. The results show that ABC triblock copolymers form smaller and more stable polyplexes with plasmid DNA than AB diblock copolymers-as verified by long-term aggregation and ethidium bromide exclusion studies-protect the DNA more effectively against nucleases, and provide better transfection efficiencies, as indicated by total protein as well as luciferase expression. More detailed studies revealed that triblock copolymers with p(L-Leu) forming the C-block were most efficient in DNA complexation with a 2.3 times higher transfection rate. Furthermore, increasing the cationic character by increasing the p(L-Lys) chain length led to up to 25% higher transfection but at the same time induced some cytotoxicity. Diblock copolymers, where the amino acid-building blocks exist as a random copolymer, bind more loosely with DNA leading to less compact and less stable aggregates with lower transfection efficiencies.