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.