Di[12]aneN3-Functionalized Green Fluorescent Protein Chromophore for GFP Luminescence Simulation and Efficient Gene Transfection In Vitro and In Vivo

Although a plethora of gene carriers have been developed for potential gene therapy, imageable stimuli-responsive gene vectors with fast access to the nucleus, high biocompatibility, and transfection efficiency are still scarce. Herein, we report the design and synthesis of four dendrite-shaped cationic liposomes, MPA-HBI-R/DOPE (R: n-butyl, 1; n-octyl, 2; n-dodecyl, 3; palmyl, 4), prepared via esterification of 4-alkoxybenzylideneimidazolinone containing aliphatic chains of different lengths (HBI-R), the green fluorescent protein (GFP) chromophore, with a di[12]aneN(3) unit. Liposomes were fabricated via the self-assembly of MPA-HBI-R, assisted with 1,2-dioleoyi-sn-glycerol-3-phosphorylethanolamine (DOPE). These liposomes (MPA-HBI-R/DOPE) exhibited efficient DNA condensation, pH-responsive degradation, excellent cellular biocompatibility (up to 150 mu M), and high transfection efficiency. Molecular docking experiments were also used to verify the optimal interaction between MPA-HBI-R and DNA, as well as the fluorescence enhancements. In particular, MPA-HBI-2/DOPE delivered DNA into the nucleus in less than an hour, and its luciferase transfection activity was more than 10 times that by Lipo2000, across multiple cell lines. The GFP chromophore conjugation allowed trackable intracellular delivery and release of DNA in real time via fluorescence imaging. Furthermore, efficient red fluorescent protein (RFP) transfection in zebrafish, with an efficiency of more than 6 times that by Lipo2000, was also achieved. The results not only realized, for the first time, the combination of gene delivery and GFP-simulated light emission, allowing fluorescent tracking and highly efficient gene transfection, but also offered valuable insights into the use of biomimetic chromophore for the development of the next-generation nonviral vectors.

Automated summary

This study reports the design and synthesis of four dendrite-shaped cationic liposomes with efficient DNA condensation, pH-responsive degradation, excellent cellular biocompatibility, and high transfection efficiency. The liposomes demonstrated fast access to the nucleus, successful gene delivery, and trackable intracellular delivery and release of DNA in real time via fluorescence imaging. The results suggest the potential of biomimetic chromophores for the development of next-generation nonviral gene vectors.