Non-viral gene transfection in vitro using endosomal pH-sensitive reversibly hydrophobilized polyethylenimine

Abstract Reversibly hydrophobilized 10 kDa polyethylenimine (PEI) based on rapidly acid-degradable acetal-containing hydrophobe was designed for nontoxic and highly efficient non-viral gene transfer. Water soluble PEI derivatives with average 5, 9 and 14 units of pH-sensitive 2,4,6-trimethoxybenzylidene-tris(hydroxymethyl)ethane (TMB-THME) hydrophobe per molecule, denoted as PEI- g -(TMB-THME)n , were readily obtained by treating 10 kDa PEI with varying amounts of TMB-THME-nitrophenyl chloroformate. Gel retardation assays showed that all PEI- g -(TMB-THME)n derivatives could effectively condense DNA at an N/P ratio of 5/1. Notably, polyplexes of PEI- g -(TMB-THME)n derivatives had smaller sizes (about 100∼170 nm) and higher surface charges (+25 ∼ +43 mV) than the parent 10 kDa PEI at the same N/P ratios ranging from 10/1 to 40/1. MTT assays revealed that these PEI- g -(TMB-THME)n derivatives were practically non-toxic at polymer concentrations used in transfection experiments. The acetal degradation of PEI- g -(TMB-THME)9 was shown to be highly pH dependent in which half lives of 1.3, 2.8 and 11 h were determined for pH 4.0, 5.0 and 6.0, respectively, while negligible hydrolysis (