Emma Gehlbach

Synthetic cells are simple, man-made objects that mimic the structure and function of living cells. They have the minimum complexity needed to sustain relevant functions and can be programmed to produce proteins through cell-free protein synthesis (CFPS). Liposomes can be used to make synthetic cells, but current methods of creating these liposomes through gentle hydration of lipids with aqueous buffer are inefficient at encapsulating aqueous solution within the liposome, which is a necessary step for making synthetic cells that can perform CFPS. Improving the encapsulation efficiency of the gentle hydration process would make it a more useful technique for synthetic cell research. This research tested whether sequentially hydrating lipids in increments of 1 mM until a final desired lipid concentration is reached leads to higher encapsulation efficiency than the conventional non-sequential process, in which all the lipids are hydrated at once. Cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids were used in the experiment since liposomes composed of both POPC and cholesterol mimic mammalian membrane lipid composition. POPC and cholesterol concentrations ranging from 1-9 mM were tested in 2 different buffer solutions: a 100 mM HEPES buffer that has applications for CFPS, and a TRIS-based CHAN buffer that is used in liposome storage. Sequential encapsulation produced significantly higher average percent encapsulation values than the non-sequential process for 4, 5, 6, and 7 mM concentrations of POPC and cholesterol in HEPES buffer. When the lipids were hydrated with CHAN buffer, the average percent encapsulation was significantly higher for the non-sequential method for 6 mM POPC and cholesterol than for the sequential process. The increase in encapsulation efficiency produced by sequential encapsulation in HEPES buffer can expand the utility of using gentle hydration to form liposomes for synthetic cell research in HEPES-based buffers.