**4. Discussion**

In recent decades, natural derivatives have been the subject of intensive studies regarding their biological and pharmacological properties. Resveratrol is known for its numerous beneficial effects on human health, even though its mechanisms of action are still unclear. Due to its lipophilicity and poor water solubility, the lipid membrane is the principal target of resveratrol.

An important aspect of resveratrol's interaction with the membrane is that it induces changes in the membrane's biophysical properties. The resveratrol molecule may induce changes either to the interfacial structure or to the chain region, thus modifying membrane packing. Therefore, it is very important to be able to clarify the exact location in or on the membrane.

In this work, by simultaneously measuring membrane conductance and capacitance, we show that resveratrol interacts with a planar lipid membrane and the mode of interaction is due to the bilayer's lipid composition. To our knowledge, this is the first study in which resveratrol's membrane activity has been monitored by electrophysiological measurements, which are not so frequently used for the characterization of drug-membrane interactions and by PLMs that complement studies performed with liposomes.

As is well known, conductance is related to the ionic current through the bilayer when a substance (a protein, peptide, or drug) incorporates into the membrane forming conductive units that span the bilayer, while capacitance is related to the property of a membrane to act as a capacitor; indeed, the lipid bilayer is an insulator separating two electrolytic media. Capacitance is directly proportional to the area of the membrane and inversely proportional to its thickness. Capacitance also depends on the characteristics of the lipids used as the insulating material.

Several studies show that the structure of lipids determines the biophysical properties of membrane (e.g., organization in bilayers, fluidity, hexagonal phases, etc.) [48]. In our work, we used POPC that is prone to form a bilayer [49,50], cholesterol that has effects on membrane fluidity, and DOPE that is prone to form inverse hexagonal phases [49,50]. During the interaction of resveratrol with the planar lipid bilayer, the variation in capacitance can help to shed light on its mechanism of action.

Our results show that resveratrol incorporates into PLMs made up of POPC and forms transient channel-like events in which lag time depends on its concentration. The lag time is the time at which a conductance variation first occurs after resveratrol addition. The different duration of lag time may be due to reaching an appropriate resveratrol/lipid ratio. Once a threshold resveratrol/lipid value has been reached, resveratrol forms transient conductive units permeabilizing the membrane. The capacitance variation, induced by adding resveratrol, may be due to: adsorption of resveratrol (capacitance decrease) occurring before formation of conductive units and/or formation of channel-like events (capacitance increase) across the membrane. The paroxystic activity reported above may be due to the collapse of the conductive units inducing rapid flip-flopping of the membrane lipids. It is important to note that both the lag time and the applied voltages at which the paroxystic activity appears depend on the resveratrol concentration, thus strengthening the concept that an appropriate resveratrol/lipid ratio has been reached.

The addition of cholesterol, a known component of the cellular membrane, to PLMs made up of POPC increases the lag time compared to that observed for experiments with POPC PLMs, regardless of the resveratrol concentration used. The capacitance behaves in a similar fashion to that observed for experiments with PLMs in the absence of cholesterol, decreasing immediately after resveratrol addition and increasing when the channel-like activity appears. Therefore, also in POPC:Ch PLMs, the formation of channel-like events comes after the resveratrol adsorption phase. However, the higher lag times than those obtained for POPC PLMs would seem to indicate that cholesterol makes it more difficult for resveratrol to incorporate into PLMs and to form conductive units, probably due to the tighter packing of the phospholipid tails.

According to some studies, the β-hydroxyl group attached to C3 of the cholesterol molecule, is located in the headgroup region of POPC, while the isooctyl chain, attached to C17, is located deep within the hydrophobic core of the bilayer close to the double bonds of fatty acids [24]. Some authors [1,25] have shown that resveratrol adopts the same position in the membrane as cholesterol. Besides, the higher applied voltage (120 mV) needed to obtain channel-like activity, compared with POPC PLMs, seems to confirm this idea.

Our results appear to be in line with those obtained by other authors [25,29,51] who have shown that resveratrol penetrates into the hydrophobic core of the bilayer close to the double bonds of polyunsaturated fatty acids.

This mode of interaction with lipids might explain the anti-peroxidation effects displayed by resveratrol, because it protects the phospholipids from oxidation reactions, acting as an electron donor to a free radical, neutralizing it and inhibiting its capacity to damage the cell membrane [52].

In contrast with its effects in neutral PLMs, resveratrol does not interact with DOPE PLMs to which negatively charged lipid DOPS have been added. To our knowledge, the interaction of resveratrol with the lipid system containing these phospholipids has never previously been studied.

In the experiments with DOPS:DOPE PLMs, the capacitance decreases after resveratrol addition reaching a minimal value that is quite constant until the end of the experiment.

The behavior of the capacitance and the absence of channel-like activity indicate that resveratrol remains on the membrane surface and is unable to incorporate into the PLM. This behavior may be due to the chemical characteristics of the lipids forming the bilayer and/or to the effect of resveratrol on the membrane surface, which promote the formation of the inverse hexagonal phase and of membrane negative curvature.

Several studies have shown that the nature and molecular shape of lipids determine their intrinsic tendency to form distinct phases [49] and to affect membrane curvature. Phosphatidylethanolamine (PE) has a truncated cone shape because its headgroup has a smaller area than the cross-section of its hydrocarbon chains, while phosphatidylcholine (PC) has a cylindrical molecular shape. Because of these features, PE is prone to form an inverse hexagonal phase and PC bilayer. Besides, dioleoyl lipids with phosphatidylethanolamine headgroups adopt a negatively-curved surface because the PE headgroup is smaller than

the PC headgroup [53], and they are able to interact most with the glycerol and phosphate groups of neighboring lipids, probably due to the differing hydration properties influencing the curvature [54].

On the other hand, it has been demonstrated that many substances that are active at the membrane interface induce membrane curvature, namely local or global deformations of the membrane.

Numerous studies show that there are many substances, interfacially active peptides [55–57] and non-peptide compounds that can influence membrane curvature, and vice versa. Yesylevskyy and colleagues have shown that the permeability of the model lipid membrane for cisplatin and gemcitabine depends on the curvature [58]. Barry and colleagues showed that curcumin, a lipophilic drug, binds to dimyristoyl-phosphocholine (DMPC) with a transbilayer orientation, anchoring to the bilayer by a hydrogen bond to the phosphate group, while, in dipalmitoleoyl-glycero-phosphoethanolamine (DiPOPE) vesicles, it promotes and stabilizes the formation of the inverted hexagonal (HII) phase. According to the authors, this feature of curcumin is indirect evidence that it induces negative membrane curvature [59].

On the other hand, primaquine, a potent antimalarial agent, increases the phase transition temperature (TH) of Lα and reduces the transition enthalpy of palmitoyl-oleoylphosphatidylethanolamine (POPE) vesicles, indicating that primaquine promotes positive membrane curvature and stabilizes the fluid phase of POPE vesicles [60].

All these studies indicate that many substances, peptides, and drugs induce modifications of a membrane's biophysical properties caused by the chemical characteristics of the substances in question. In line with this concept, our work shows that resveratrol interacts with model membranes, modifying their biophysical parameters for different lipid compositions. Studies on drug-membrane interaction are very important in order to understand and clarify their mechanism of action.
