Pressure Induced Fusion (Pif) Liposomes: A Solventless Sterilizing Method for Producing Large Phospholipid Vesicles

Abstract

We have recently described a new method to create liposomes of high captured volume we term interdigitation-fusion vesicles or IFVs (Ahl, et al. (1994) Biochim. Biophys. Acta 1195: 237–244). These vesicles arise from small (<100 nm) precursor liposomes comprised of saturated chain phospholipids which fuse upon induction to the interdigitated gel state after ethanol addition to form extensive sheets. When heated to the liquid crystalline state, these sheets vesicularize into large, predominantly unilamellar liposomes (IFVs). Here we report that a similar event occurs when hydrostatic pressures capable of causing acyl chain interdigitation are applied to small precursor vesicles of DPPC or DSPC. When DPPC small unilamellar vesicles were cycled three times between ambient pressure and 4.1 kbar (15 minute intervals) the resulting pressure induced fusion (PIF) vesicles had captured volumes of 13.1 ± 0.6 μl/μmole and were unilamellar as judged by ³¹P-NMR. Although three cycles yielded the maximum captured volume for DPPC, six pressurization cycles were required for DSPC but resulted in PIF vesicles of greater captured volume (27–37 μl/μmole). Conditions which interfere with bilayer interdigitation and have been shown to compromise IFV formation (i.e., cholesterol addition or the presence of cìs unsaturation) also compromised PIF vesicle formation. Significantly, temperatures and pressures ideal for PIF formation (40–60°C and 3.4–6.2 kbar) completely eradicated B, subtilis and B. stearothermophilus spores. The PIF procedure will be useful in a variety of biological and pharmacological applications because it avoids the use of organic solvent and allows sample sterilization at relatively low temperatures.