Effects of POPG and Cholesterol on Freeze–Thawing Mediated Content Exchange and Transcription in Giant Unilamellar Vesicles

Freeze–thaw (F/T)‐induced content mixing between artificial cells enables efficient exchange of encapsulated components. However, F/T treatment is often accompanied by vesicle rupture and content loss, and the effects of membrane composition on fusion and reaction efficiency remain unclear. Here, we investigated how cholesterol and the negatively charged phospholipid 1‐palmitoyl‐2‐oleoyl‐ sn ‐glycero‐3‐phospho‐(1′‐ rac ‐glycerol) (POPG) influence giant unilamellar vesicle (GUV) stability, content mixing, and transcriptional activity after F/T treatment. Cholesterol‐containing GUVs exhibited increased vesicle loss and reduced content mixing after F/T treatment. In contrast, POPG markedly improved GUV recovery and suppressed vesicle loss, even in the presence of cholesterol. Membranes containing 20 mol% POPG and 20 mol% cholesterol maintained high cargo retention and efficient content mixing. When DNA‐containing liposomes were fused with transcription‐reagent‐containing liposomes through F/T treatment, this membrane composition also yielded the highest transcriptional activity. Notably, although the combination of POPG and cholesterol inhibited transcription in intact GUVs, it showed the highest transcriptional activity following F/T‐mediated content mixing. These findings demonstrate that the effects of membrane composition on encapsulated biochemical reactions depend strongly on whether the reaction components are initially coencapsulated or brought together through F/T‐mediated content exchange.