Novel symmetric amphiphilic dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic poly(L-lysine) with high plasmid DNA binding affinity as a biodegradable gene carrier

This study communicates the molecular design, preparation, and biological application of novel symmetric amphiphilic polycationic dendritic poly(L-lysine)-b- poly(L-lactide)-b-dendritic poly(L-lysine) D-2-LLA(15)-D-2 bearing two two-generation poly(L-lysine) PLL dendrons D-2 and a central hydrophobic biodegradable poly(L-lactide) block LLA(15). First, an amino-protected precursor of L-1- OH was designed and synthesized and was further employed to prepare L-1-LLA(15) with an organic 4-(dimethylamino)-pyridine-mediated living-ring-opening polymerization of L-lactide. Subsequently, the hydroxy end-capped L-1-LLA(15) was coupled to synthesize a new triblock L(1)LLA(15)- L-1 with two one-generation amino-protected PLL dendrons L-1. Furthermore, with a repeated trifluoroaceticacid-mediated amino deprotection-protection cycle, new amphiphilic triblock D-2-LLA(15)-D-2 was successfully prepared. By means of NMR, mass spectrometry, and gel permeation chromatography, these synthetic precursors and final amphiphilic product were characterized to bear well- defined triblock structures. In addition, this synthesized amphiphilic triblock polycationic macromolecule was applied as a new polycationic plasmid DNA carrier, and its DNA binding affinity was examined via an agarose electrophoresis and a fluorescence titration assay along with two important references of hydrophilic dendritic D-2-HEX-D-2 and double- hydrophilic D-2-PEG- 4K-D-2 bearing the same two D-2 dendrons; much enhanced DNA binding affinity was interestingly revealed for the new amphiphilic structural D-2-LLA(15)-D-2. Moreover, the assembled polyplex microparticles of plasmid DNA/polycationic carrier were further analyzed by dynamic light scattering and transmission electron microscopy, indicating their averaged nanoparticle size around 150-200 nm. As for the cytotoxicity of the new D-2- LLA(15)-D-2, MTT assays were conducted with a human hepatocellular carcinoma cell line (SMMC-7721), indicating a very low cytotoxicity as compared with commercial linear PLL-23K and PEI-2K, and a DNase I degradation of the assembled polyplex particles was also done in the HBS buffer solution to evaluate their stabilities. Finally, employing the new amphiphilic D-2-LLA(15)-D-2 as gene carrier, in vitro gene transfection experiments were conducted with the SMMC-7721 cell line, indicating a transfection efficiency increase of at least 10 times higher than that of the naked plasmid DNA under a N/P charge ratio of 10. Therefore, these interesting results may provide a new possible way to construct efficient polycationic macromolecular gene carriers with low toxicity and less expensive low-generation PLL dendrons.