Branched Polyethylenimine Derivatives With Reductively Cleavable Periphery For Safe And Efficienct In Vitro Gene Transfer

Gene therapy is a broad term that encompasses any strategy to treat a disease by introducing an exogenous gene into cells of an affected person. The safe and effective delivery of therapeutic genes remains a critical bottleneck in the field of gene therapy. Although viral vectors have demonstrated high transfection efficiencies, their safety, sophisticated production and limited gene-encapsulation capacity remain issues to be solved. Therefore, non-viral synthetic carriers have received more and more attention, because of their many advantages over viral vectors, such as improved safety, ease of large-scale production, possible repeated administration, and facile vector modification. One of the most successful and widely studied gene delivery polymers reported to date is polyethylenimine (PEI). Due to its relatively high gene delivery efficiency, 25 kDa branched PEI has become a golden standard for newly designed polymeric gene carriers. However, 25 kDa branched PEI has lower transfection efficiency compared to viral systems and demonstrates rather high cytotoxicity.In this study, novel 25 kDa PEI derivatives with reductively cleavable cystamine periphery (PEI-Cys) were designed to reduce carrier-associated cytotoxicity and to further enhance the transfection activity. The Michael-type conjugate addition of 25 kDa PEI with N-tert-butoxycarbonyl- N′-acryloyl-cystamine (Ac-Cys-t~Boc) and N-tert-butoxycarbonyl- N′-methacryloyl-cystamine (MAc-Cys-t~Boc) followed by deprotection readily afforded PEI-Cys derivatives, denoted as PEI-(Cys)_x(Ac) and PEI-(Cys)_x(MAc), with degree of substitution (DS) ranging from 14 to 34 and 13 to 38, respectively. All PEI-Cys derivatives had higher buffer capacity than the parent 25 kDa PEI (21.2-23.1 % versus 15.1 %). Gel retardation assays showed that cystamine modification resulted in enhanced interaction with DNA. PEI-(Cys)x(Ac) could condense DNA into small-sized particles of 80-90 nm at and above an N/P ratio of 5/1, which were smaller than polyplexes of 25 kDa PEI (100-130 nm). In comparison, PEI-(Cys)_x(MAc) condensed DNA into somewhat larger particles (100-180 nm at N/P ratios from 30/1 to 5/1). Gel retardation and dynamic light scattering (DLS) measurements showed that PEI-Cys polyplexes were quickly unpacked to release DNA in response to 10 mM dithiothreitol (DTT). These PEI-Cys derivatives revealed markedly decreased cytotoxicity as compared to 25 kDa PEI (IC50: > 100 mg/L versus ca. 11 mg/L). The in vitro transfection experiments in HeLa and 293T cells using pGL3 as a reporter gene showed that gene transfection activity of PEI-Cys derivatives decreased with increasing DS and PEI-(Cys)_x(MAc) exhibited higher transfection activity than PEI-(Cys)_x(Ac) at similar DS. Notably, polyplexes of PEI-(Cys)14(Ac) and PEI-(Cys)13(MAc) showed significantly enhanced gene transfection efficiency (up to 4.1-fold) as compared to 25 kDa PEI formulation at an N/P ratio of 10/1 in both serum-free and 10 % serum-containing conditions. The modification of PEI with reductively cleavable periphery appears to be a potential approach to develop safer and more efficient non-viral gene vectors.