Synthesis of Novel Biodegradable Quaternary Amine-based Cross-linked Poly(β-amino ester) and Its Self-assembly/disassembly with Plasmid DNA

Abstract

In recent years, various cationic polymers constructed using ester bonds, phosphoester bonds or disulfide bonds have been synthesized to create environmentally-responsible biodegradable delivery systems. Among them, a crosslinked type polymeric gene delivery carrier was reported to show enhanced transfection efficiency due to its threedimensional structure leading endosome buffering effect, and self-assemble with plasmid DNA giving more stable polyplexes due to its slow degradation in aqueous media, in contrast to linear type carriers. Several quaternary amine-based polymeric gene delivery carriers were reported and they showed considerable gene delivery potencies. Moreover, a primary amine-based biodegradable polymeric carrier was known to have potentially rapid self-destructive tendency due to the attack of its own primary amines. Because quaternary amines have no nucleophilicity, quaternary amine-based biodegradable polymeric carriers have no possibility of ‘self-destruction’. So, we designed a novel quaternary amine-based crosslinked poly(β-amino ester) polymer (Q-CLPAE). Here, we report the synthesis, characterization of QCLPAE. In addition, the self-assembly of polymers with plasmid DNA and in vitro disassembly of plasmid DNA from polyplexes are presented. Q-CLPAE was synthesized by using a Michael reaction of a quaternary amine monomer and a triacrylate monomer according to Lynn et al. First, a quaternized amine monomer was synthesized through the following steps (Scheme 1). Fmoc blocking reaction of N,N-dimethylethylenediamine (DMEDA). Fmoc-OSu (1 g, 2.96 mmol, Novabiochem, Laufelfingen, Switzerland) was dissolved in 1,4dioxane. DMEDA (0.488 mL, 4.45 mmol, Sigma-Aldrich, St. Louis, MO) was dissolved in 1,4-dioxane and added to an Fmoc-OSu solution dropwise, keeping it at r.t. overnight. Then, the reaction mixture was poured into diethyl ether and extracted with water to remove the residual DMEDA. The ether layer was extracted again with 0.1 M HCl solution. At this time, the acidified product was transferred to the HCl layer and the residual Fmoc-OSu was left in the ether layer. The HCl layer was basified to ~ pH 11 with 1 N NaOH solution and the hydrophobic product in the aqueous layer was extracted with diethyl ether. Finally, after evaporation of the ether layer, a purified product, N-(9-Fluorenylmethoxycarbornyl)-N',N'-dimethylethylenediamine (Fmoc-DMEDA) was left as a white solid. Methylation of Fmoc-DMEDA. Fmoc-DMEDA (195 mg, 0.598 mmol) was dissolved in DMF. 3.72 mL of iodomethane (d = 2.28, 59.8 mmol, Sigma-Aldrich) was added to an Fmoc-DMEDA solution, keeping it at r.t. overnight. The product was precipitated with diethyl ether three times to remove the unreacted materials, leaving a light yellow solid, N-(9-Fluorenylmethoxycarbornyl)-N',N',N'trimethylethylenediamine (Fmoc-TMEDA). Fmoc deblocking of Fmoc-TMEDA. Fmoc-TMEDA (101 mg, 0.296 mmol) was dissolved in DMF and mixed with an equal volume of 30% piperidine solution (DMF). After stirring at room temperature, the product was pre-