Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

Xiao, Qin; Liu, Yuheng; Zhang, Dandan; Li, Chao; Yang, Qihua; Lu, Dongshui; Zhang, Weijun; Rosenecker, Joseph; Zou, Quanming; Li, Yang und Guan, Shan (2022): Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles. In: Jove, Nr. 186

Abstract

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp is produced by mixing synthetic peptide/ poloxamine with mRNA solution using a microfluidic mixer, allowing for the selfassembly of PP-sNp. The charge of PP-sNp is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PPsNp nanoparticles are measured using dynamic light scattering. The mRNA/PPsNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing nextgeneration mRNA vaccine delivery systems.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Medizin
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• ISSN: 1940-087X
• Sprache: Englisch