Protocol for formulation and evaluation of phytosterol-based lipid nanoparticles as cholesterol alternatives for mRNA delivery

We present a protocol for formulating and evaluating lipid nanoparticles (LNPs) that incorporate the plant-derived sterols ginsenoside Rg2 and protopanaxadiol (PPD) as substitutes for cholesterol. The protocol describes mRNA preparation, manual mRNA-LNP encapsulation, and physicochemical characterization. We further detail in vitro and in vivo procedures to evaluate mRNA delivery efficiency. This protocol provides a robust and cost-effective platform for efficient mRNA delivery. For complete details on the use and execution of this protocol, please refer to Park et al. 1 • Formulation of LNPs using plant-derived sterols Rg2 and PPD instead of cholesterol • Manual workflow for mRNA synthesis, encapsulation, and analysis • Demonstrates efficient mRNA delivery using Rg2-LNP and PPD-LNP in vitro and in vivo • Provides optimization guidance for cell-type-specific formulations Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. We present a protocol for formulating and evaluating lipid nanoparticles (LNPs) that incorporate the plant-derived sterols ginsenoside Rg2 and protopanaxadiol (PPD) as substitutes for cholesterol. The protocol describes mRNA preparation, manual mRNA-LNP encapsulation, and physicochemical characterization. We further detail in vitro and in vivo procedures to evaluate mRNA delivery efficiency. This protocol provides a robust and cost-effective platform for efficient mRNA delivery.