The impact of carboxyalkylation of branched polyethylenimine on effectiveness in small interfering RNA delivery

Abstract

Carboxyalkylation of branched 25 kDa polyethylenimine (PEI) was considered to reduce the positive surface charge of the polymer without reducing its 'proton sponge' buffering capacity, and to provide alkylene domains for hydrophobic interactions, thus generating optimized novel PEI carriers for efficient delivery of small interfering RNA (siRNA). Substitution of PEI was evaluated in the range of 6 to > 50 mole percentage of primary amines. Additionally, variation of the carboxyalkyl chain (one to 15 methylene groups) was explored to modulate the carrier hydrophobicity. Carriers were characterized in their buffering capacity, capability of siRNA polyplex formation, and cytotoxicity. Marker gene-silencing efficacy was evaluated using Neuro2A-eGFPLuc neuroblastoma cells. Carboxyalkylation strongly reduced cytotoxicity of PEI and improved siRNA mediated luciferase gene knockdown. An optimum silencing activity was observed at an alkylcarboxylation degree of 6-9 mole percentage of primary amines and with a broad range of carboxyalkylene chains (containing one to 15 methylene groups). Strongly enhanced gene-silencing efficacy also was observed when the biocompatible polymers were separately added at 1 h after transfection with tolerated doses of standard PEI25/siRNA polyplexes. Carboxyalkylation of branched 25 kDa PEI resulted in polymers with strongly reduced cytotoxicity and improved silencing efficacy. Mechanistic studies demonstrated that the presence of a surplus of free carboxyalkylated polymer is responsible for the improved siRNA delivery.