High performance mRNA transfection through carbonate apatite-cationic liposome conjugates.
mRNA instead of DNA provides a new and attractive approach for gene therapy and genetic vaccination. Delivery of mRNA can bypass nuclear localization step enabling protein expression directly in cytoplasm through transcription. Current technologies for mRNA delivery are predominantly based on cationic liposomes with low activity for transfection. We, previously reported that applying inorganic nano-particles of carbonate apatite onto cationic liposome of DOTAP {N-[1-(2,3-dioleoloxy)propyl]-N,N,N-trimethyl ammonium chloride} resulted in high transfection potency for luciferase mRNA both in mitotic and non-mitotic cells. In this paper, we expanded the previous work and performed in detail study especially on two important parts, evaluating the image of the complex and analyzing the steps of gene delivery to detect the determinant factor for enhanced transfection potency. Transmission electron microscopic (TEM) observation clearly indicated the presence of inorganic carbonate apatite particles on mRNA-liposome complex and demonstrated the structure of the new hybrid carrier material. Due to apparently higher gravitational force of absorbed inorganic nano-particles, cellular contact and internalization of hybrid-particle-associated mRNA were significantly enhanced compared to DOTAP. This analysis indicates rather than downstream steps, initial steps of cell membrane binding and subsequent way of internalization could be the determinant factor for final protein expression. Moreover, we compared transfection efficiency of mRNA and pDNA in Human Umbilical Vein Endothelial cell (HUVEC) to demonstrate advantages of mRNA delivery.
Zohra
Fatema T
FT
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Chowdhury
Ezharul H
EH
Akaike
Toshihiro
T
D001031:Apatites / Q000737:chemistry*; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D012333:RNA, Messenger / Q000008:administration & dosage*; D014162:Transfection / Q000379:methods*
D001031:Apatites; D008081:Liposomes; D012333:RNA, Messenger; C030782:carboapatite