A reducible polycationic gene vector derived from thiolated low molecular weight branched polyethyleneimine linked by 2-iminothiolane

Abstract

To improve transfection efficiency and reduce the cytotoxicity of polymeric gene vectors, reducible polycations (RPC) were synthesized from low molecular weight (MW) branched polyethyleneimine (bPEI) via thiolation and oxidation. RPC (RPC-bPEI 0.8kDa ) possessed a MW of 5 kDa~80 kDa, and 50%~70% of the original proton buffering capacity of bPEI 0.8kDa was preserved in the final product. The cytotoxicity of RPC-bPEI 0.8kDa was 8~19 times less than that of the gold standard of polymeric transfection reagents, bPEI 25kDa . Although bPEI 0.8kDa exhibited poor gene condensing capacities (~2 µm at a weight ratio (WR) of 40), RPC-bPEI 0.8kDa effectively condensed plasmid DNA (pDNA) at a WR of 2. Moreover, RPC-bPEI 0.8kDa /pDNA (WR ≥ 2) formed 100~200 nm-sized particles with positively charged surfaces (20~35 mV). In addition, the results of the present study indicated that thiol/polyanions triggered the release of pDNA from RPC-bPEI 0.8kDa /pDNA via the fragmentation of RPC-bPEI 0.8kDa and ion-exchange. With negligible polyplex-mediated cytotoxicity, the transfection efficiencies of RPC-bPEI 0.8kDa / pDNA were approximately 1200~1500-fold greater than that of bPEI 0.8kDa /pDNA and were equivalent or superior (~7-fold) to that of bPEI 25kDa /pDNA. Interestingly, the distribution of high MW RPC-bPEI 0.8kDa /pDNA in the nucleus of the cell was higher than that of low MW RPCbPEI 0.8kDa /pDNA. Thus, the results of the present study suggest that RPC-bPEI 0.8kDa has the potential to effectively deliver genetic materials with lower levels of toxicity.

The effect of environmental pH on polymeric transfection efficiency

“…A great effort for developing effective polymeric vectors has focused primarily on cellular receptor targeting [1-3], endosomal escape [4-6], cytosolic transport [7-9], nuclear import [8, 10, 11], and decomplexation [12-17]. The effects of the transfection environment with respect to proteins [18-20], ions [3, 18, 21-23], pH [13, 16, 17, 24, 25], reduction/oxidation potentials [14, 15, 26], and hypoxia [14, 27-29] have also been investigated in relation to polyplex preparation and polymeric transfection.…”

Acid-Labile Poly(glycidyl methacrylate)-Based Star Gene Vectors

“…The interaction of cationic vectors and basic components (cell membrane and proteins) is an important cause to cell apoptosis. 41 As reported earlier, hydroxyl groups in the repeat units of PGEAs played an important role in shielding effect for detrimental excess positive charge. 17−20 A-CD-PGEAs and CD-PGEAs with different molecular weights were much less toxic than "gold standard" PEI at varied N/P ratios in COS7, C6, and HeLa cell lines (Figure 8).…”

Reducible chimeric polypeptide consisting of octa-d-arginine and tetra-l-histidine peptides as an efficient gene delivery vector

“…Compared with DMSO, which is widely used in the study of reducible polymers, 16,20,22 the oxidative reaction of thiol groups using low-concentration H 2 O 2 in the present study was relatively mild without producing by-products. The reaction conditions were optimized for the best transgene expression ability.…”

“…20,21 In this study, we synthesized a series of reducible chimeric polypeptides known as RHss that contained octa-d-arginine and tetra-l-histidine peptides with various ratios. After physical characterization and transfection efficiency evaluation, we screened out a Abbreviations: he, hematoxylin-eosin; PBs, phosphate-buffered saline; pGL3, plasmid encoding luciferase gene; pTRAIL, plasmid encoding tumor-necrosis-factor-related apoptosis-inducing ligand; rhss, reducible chimeric polypeptide containing two functional peptides: octa-d-arginine and tetra-l-histidine peptides; rlU, relative luciferase unit; rss, single cross-linked r8 peptide; sD, standard deviation.…”