Post-modification of poly(glycidyl methacrylate)s with alkyl amine and isothiocyanate for effective pDNA delivery

Abstract

Thiourea has been shown to interact actively with phosphate groups of DNA. To this end, linear and five-arm poly(glycidyl methacrylate)s (PGMA)s are post-modified with 1,2-ethanediamine (E), 1,4-butanediamine (B) and diethylenetriamine (D) via ring opening reactions to obtain various amino poly(glycidyl methacrylate)s (PGOHMAs), namely L–E, S5–E, S5–B, L–B, and L–D (L represents linear polymer, S5 represents five-arm polymer, E is short for 1,2-ethanediamine, B is short for 1,4-butanediamine, and D is short for diethylenetriamine). These amino PGOHMAs are further functionalized with methyl isothiocyanate (M) to give thiourea-modified PGOHMAs (TPGOHMAs), L–EM, S5–EM, L–DM, L–BM, S5–BM, L–EM and S5–EM, with propyl isothiocyanate (P) to give L–BP. Both the amino PGOHMAs and TPGOHMAs are used as gene vectors. The mean hydrodynamic diameter of amino PGOHMA/plasmid DNA (pDNA) and TPGMP/pDNA complexes is in the range of 65–195 nm, as determined by dynamic light scattering, and decreases with increasing nitrogen-to-phosphate (N/P) ratio. At the same N/P ratio, TPGOHMA/pDNA complexes exhibit lower values of zeta potentials than its amino PGOHMA counterpart when zeta potentials turned into positive. Gel electrophoresis indicates that TPGOHMA polymers can condense pDNA at lower N/P ratios than amino PGOHMAs. MTT assay suggests that TPGOHMAs have obviously lower cytotoxicity than amino PGOHMAs against Huh-7 cells. Subsequently, in vitro gene transfection studies against Huh-7 cells show that TPGOHMA/pDNA exhibits lower transfection efficiency than amino PGOHMAs, except for L–DM/pDNA complexes. Confocal laser scanning microscopy reveals that L–DM/pDNA complexes achieve endosomal escape more effectively than L–D/pDNA complexes. This study revealed the effect of thiourea-modification of amino PGOHMA on its gene transfection properties. L–DM, with enhanced transfection efficiency and reduced cytotoxicity, is an interesting material for further investigation as a nucleic acid delivery vehicle.