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

摘要: 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 14 units of pH-sensitive 2,4,6-trimethoxybenzylidene-tris(hydroxymethyl)ethane (TMB-THME) per molecule, denoted as PEI-g-(TMB-THME)(n), were readily obtained by treating varying amounts TMB-THME-nitrophenyl chloroformate. Gel retardation assays showed that all PEI-g-(TMB-THME)(n) could effectively condense DNA at an N/P ratio 5/1. Notably, polyplexes had smaller sizes (about 100∼170 nm) higher surface charges (+25 ∼ +43 mV) than the parent same ratios ranging from 10/1 to 40/1. MTT revealed these practically non-toxic polymer concentrations used in transfection experiments. The acetal degradation PEI-g-(TMB-THME)(9) shown be pH dependent which half lives 1.3, 2.8 11 h determined 4.0, 5.0 6.0, respectively, while negligible hydrolysis (<12%) observed after 24 7.4. electrophoresis, dynamic light scattering (DLS) zeta potential analyses indicated formed dissociated following 5 incubation 5.0, highlighting importance hydrophobic TMB-THME moieties condensation supporting endosomes would facilitate release. vitro experiments performed 20/1 HeLa, 293T, HepG2 KB cells using plasmid pGL3 expressing luciferase reporter reversibly PEIs superior activity 25 control. For example, PEI-g-(TMB-THME)(14) about 235-fold 175-fold efficiency compared HeLa serum-free 10% serum media, approximately 7-fold 16-fold formulation its optimal under otherwise conditions. Confocal laser scanning microscope (CLSM) studies confirmed efficiently delivered Cy5-labeled nuclei cells. These endosomal have great potentials safe transfection.