*3.4. Resveratrol Interaction with DOPS:DOPE PLMs*

In order to test the role of the polar head and the effect of negative membrane charge on resveratrol incorporation and channel-like events, we carried out experiments using PLMs made up of DOPS:DOPE (1:2, *w*/*w*) in which the negative charge is diluted. This membrane is characterized by the presence of the net negative charge of DOPS and the neutral DOPE, a non-lamellar-forming lipid.

The resveratrol concentrations tested were 10 and 20 µM. The addition of resveratrol to the *cis* side of the medium facing the membrane was made at an applied voltage of 100 mV, using the same protocol as that described for POPC and POPC:Ch PLMs.

In all experiments with DOPS:DOPE membranes and independently of the resveratrol concentration, no current fluctuations were observed for many hours using a wide range of applied voltages. No channel-like activity occurred after PLM breakage and the formation of the second membrane by painting the lipid solution present around the hole.

In addition, in these experimental sets, we monitored capacitance. In all experiments, before the addition of resveratrol, the basic capacitance of the PLMs remained constant in the range of 0.26–0.28 µF/cm<sup>2</sup> . After the addition of resveratrol, we observed that the

capacitance decreased during the lag time until reaching a minimum value that remained constant until the end of the experiment. This behavior was observed both at 10 and 20 µM of resveratrol. As in the experiments with POPC and POPC:Ch PLMs, we calculated the mean capacitance values at the different times, T0, T1, T2, and T3, which are reported in Table 5. The capacitance values remain low (0.11 <sup>±</sup> 0.03 <sup>µ</sup>F/cm<sup>2</sup> ) even after PLM breakage and formation of the second membrane.

**Table 5.** Capacitance variation in DOPS:DOPE PLM. Mean values of the membrane capacitance (C ± SE) calculated at T0, T1, T2, and T3, for which meanings are reported in the legend to Table 3. The mean value was obtained from at least six experiments.


These results indicate that resveratrol is unable to incorporate into PLMs made up of DOPS:DOPE and to form channel-like events, probably due to the presence of DOPE, a nonlamellar-forming lipid, as resveratrol may induce a negative curvature of the membrane. The combination of these factors prevent resveratrol incorporation into PLMs; however, we cannot exclude an effect of negatively charged lipid DOPS.
