Quant ittm ribogreen assay

Ionizable LNPs were kindly provided by EnhancedBio Inc. (Seoul, South Korea). LNPs were prepared using the NanoAssemblr Benchtop Instrument (Precision Nanosystems Inc., Vancouver, Canada) according to a previously described method [28 (link)]. The lipid components (ionizable lipid, distearoylphosphatidylcholine, cholesterol, and polyethylene glycol lipid at a molar ratio of 42.5:13:43.5:1.0) were dissolved in ethanol, and HPV16 E6/E7 siRNAs (target sequence: 5’-GAC CGG UCG AUG UAU GUC UUG-3’) were dissolved in 50 mM sodium acetate. The final weight ratio of ionizable lipid to RNA was 7.5:1, and the final volume ratio was 1:3. LNPs were formulated by microfluidic mixing of the prepared solutions at a flow rate of 12 ml/min. The resulting LNPs were dialyzed against 1X phosphate-buffered saline (PBS) using dialysis cassettes with a 10,000 molecular weight cutoff (Life Technologies, CA, USA) for 16 hours to exchange the buffer. To characterize the prepared LNPs, dynamic light scattering was used to confirm their size, polydispersity index (PDI), and zeta potential. The encapsulation efficiency of RNAs was measured using the Quant-iTTM Ribogreen Assay (Life Technologies, CA, USA).