PS-GAMP synthesis

1. All lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Avanti Polar Lipids), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG, Avanti Polar Lipids), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000, Avanti Polar Lipids) and Cholesterol (Sigma Aldrich) were mixed at a mass ratio of 10:1:1:1 in 3 ml of chloroform (Sigma Aldrich). 

2. Chloroform was then mixed with 1 ml cGAMP solution (200 µg cGAMP, 13.7 mM NaCl, 0.27 mM KCl, 0.43 mM Na₂HPO4, and 0.147 mM KH₂PO4). 

3. The liposomes were synthesized by reverse-phase evaporation. The mixture of lipids and cGAMP was sonicated to achieve a water-in-oil emulsion under N₂ for 30 min at 50°C. 

4. Remove the solvent via rotary evaporation (Buchi) at a speed of 220 rpm. 

5. An excess amount of buffer (13.7 mM NaCl, 0.27 mM KCl, 0.43 mM Na₂HPO4, and 0.147 mM KH₂PO4) was added to the mixture and continuously rotated for another 5 min at 50°C. 

6. Resultant liposomes were extruded through 400- and 200-nm membranes (Avanti Polar Lipids) at 50°C. 

7. The size and zeta potential of liposomes were measured by Zetasizer (Malvern). 

8. Free cGAMP was removed by a size-exclusion column G-50 (GE Healthcare). 

9. To stabilize the liposomes, Trehalose (Sigma Aldrich) was added to the liposome suspension at a final concentration of 2.5%. 

10. The resultant suspension was frozen in dry ice/ethanol bath and then lyophilized at −45°C under vacuum by Freezone 4.5 (Labconco). 

11. The lyophilized liposome (PS-GAMP) was stored at −20°C.