Phase Separation and Passive Diffusion of Perfluorooctane Sulfonic Acid (PFOS) in Multilamellar and Unilamellar Vesicles

Abstract

Perfluorinated alkyl substances (PFAS) are important environmental hazards that enter microorganisms and animal tissues via their cellular membranes, where they bind to both proteins and lipids1. The interaction of a prevalent PFAS, perfluorooctane sulfonic acid (PFOS), with a model cell membrane composed of dipalmitoyl phosphatidylcholine (DPPC) was investigated as a function of molar ratio of DPPC/PFOS in both multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs). The PFOS was both prepared and incubated with the vesicles and its incorporation into the LUVs and MLVs was monitored by nano- differential scanning calorimetry (for phase transition temperatures, Tm) and by dynamic light scattering (DLS) or optical microscopy for size. For MLVs and LUVs prepared with PFOS, no pretransition was observed. The LUVs and MLVs remained intact for up to 30 days with sizes ~ 100nm for LUVs and ~ 10-100 μm for MLVs. At DPPC/PFOS ~ 75/1 to 7.5/1, there was a single Tm, that decreased and broadened as the DPPC/PFOS molar ratio decreased, as previously observed.2 At higher PFOS concentrations, DPPC/PFOS < 5/1, two or three phase transitions were observed, with one Tm at a temperature close to that of the neat MLVs/LUVs and one at lower temperature. This was interpreted as phase separation into PFOS rich and PFOS poor domains. When MLVs were incubated with PFOS, both the main (Tm) and pretransition (Tpre), characteristic of neat DPPC, were observed, indicating the presence of bilayers with no incorporated PFOS. The intensity of Tm decreased with increased time, temperature (i.e. faster above than below Tm) and the external PFAS concentration, and Tpre increased (T = Tm - Tpre decreased). Concurrently, a phase transition in the MLVs at lower temperature was observed and disappeared with time. These results indicate that there was progressive penetration of the PFOS from the outer leaflets (that had incorporated PFOS) to the interior bilayers (that had no incorporated PFOS) of the MLVs, and by implication that there was passive diffusion of PFOS across (not just into) the DPPC bilayers, which occurred more quickly above than below Tm. While diffusion of PFOS across cellular membranes has previously been observed, this effect has been attributed to association with membrane proteins.