Preparation of LNP-IL-21 particles
Lipid nanoparticle formulations were prepared using the microfluidic
mixing step as previously described [15]. Briefly, lipids were
dissolved in ethanol containing cationic lipids (ALC-0315),
lipid-anchored PEG-2000, cholesterol and disaturated phosphatidyl
choline (DSPC) with molar ratios of 46.3: 1.6: 42.7: 9.4. The IL-21 mRNA
was dissolved in 10 mM citrate buffer (pH = 4) and combined with above
lipid mixture. The ethanol phase and aqueous phase were then mixed using
a microfluidic mixer INanoL (Micro & Nano Technology Co. Ltd.,
Shanghai, China) at a flow rate ratio of 1 to 3 with the total flow rate
of 12 ml/min. The LNP formulations were then diafiltrated using
ultrafiltration centrifugal tube (Millipore, 15ml/10kD). The samples
were then collected, diluted with 1 X PBS and stored at 4℃ for further
analysis.