The Effect of Cholesterol on the Electroporation Process

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The goal of this work is to better understand the pore formation mechanisms during electroporation, a technique that uses an electric field to transiently permeabilize membranes and introduce foreign material such as genes into cells. We investigate the contribution of different components found in cell membranes to this process by using planar models of cell membranes whose composition is easily varied, and focus here on cholesterol. Pore formation is studied by measuring the electroporation threshold (Vth), the potential at which pores appear in the membranes. We start with simple mono-phospholipid membranes to which cholesterol is added. Cholesterol stabilizes DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) membranes until 10% cholesterol (Vth of 255 mV vs. 200 mV without cholesterol) and subsequently strongly destabilizes it (100 mV at 35%). With L-α-PC (L-α-phosphatidylcholine) an opposite trend is observed: a slight destabilization below 20% cholesterol (Vth decrease from 165 to 110 mV) followed by a marked stabilization (420 mV at 50%). This concentration- and phospholipid-dependent effect is in good agreement with other reports and can be correlated to the molecular organization in the membrane and its thickness. However, cholesterol preferably interacts with saturated sphingolipids in natural membranes to form liquid-ordered microdomains. Sphingolipids are consequently taken into account, and modeled with sphingomyelin (SM). Sphingomyelin does not interact with L-α-PC, and strongly destabilizes L-α-PC membranes (Vth of 50 mV for 85% SM). Conversely, the addition of cholesterol totally changes the properties of membranes prepared from various L-α-PC:SM ratios: we observe a step-wise stabilization of the membrane that can be correlated with the presence of different phases (gel, liquid-ordered and liquid-disordered) in the membrane. We are currently investigating other ternary systems to better understand the combined role of these three constituents on the process of pore formation.