**4. Discussion**

Cinnamic acid and its derivatives have attracted the attention of more and more researchers due to the broad variety of pharmaceutical and biological activities and due to the wide technological or industrial applications [50]. Polyphenols are important primarily because of their antioxidant property, antitumor activity or hypertension-preventing effect. Interactions of these phenolic acids with cell membranes play an essential role in their transport, distribution, selectivity, activity, and toxicity.

Small organic acids such as hydroxycinnamic acids have been postulated to interact with/and penetrate into biological membranes, however, their ability to demonstrate such an interaction with lipid membranes can be greatly influenced by the type of substituents attached to the main structure. Therefore, structurally similar compounds may differently interact with biological membranes, because of the complex relationship (which can be modulated by pH changes) between liposolubility and permeability [51]. The dissociation constant is a very important parameter describing solubility, lipophilicity, and permeability properties of compounds. Phenolic acids in aqueous solutions exhibit protonated and deprotonated forms. Analyzing the values of dissociation constants of the carboxyl and hydroxyl groups of the studied phenolic acids (Table 7), it can be stated that their molecular and monodeprotonated forms prevail within the range of physiological pH values. Moreover, the corresponding dissociation constant values are all similar to each other, which demonstrates that they are not a main factor determining the activity of the hydroxycinnamic acids. However, these data could be useful in studies concerning bioavailability and pharmacokinetics of potential pharmacological agents [52].

**Table 7.** Basic physicochemical parameters of cinnamic acid and its derivatives.


<sup>1</sup> molecular weight, <sup>2</sup> dissociation constant of the carboxyl groups, <sup>3</sup> dissociation constant of the hydroxyl groups, 4 calculated n-octanol/water partition coefficient, <sup>5</sup> distribution coefficient at pH 5.2 <sup>6</sup> hydrogen bond acceptor groups, <sup>7</sup> bond donor groups.

The positive values of the calculated log*P* of CinA, *p*-CoA and FA may indicate that these acids are partitioning in the octanol phase. Nevertheless, only lipophilicity of uncharged molecules can be determined by this method, thus it is difficult to apply to these phenolic acids, which at physiological conditions are rather negatively charged. Analysis of Table 7 revealed that there are relevant differences between *c*log*P* and log*D* coefficient partitions for each acid. These findings suggest that the drug distribution between the aqueous solution and the lipid membrane is regulated by the ionization state of the molecule. All three compounds tested here are ionizable molecules, and it is already widely recognized that ionizable drugs partition into the lipid membrane to a high extent thanks to electrostatic interactions and formation of hydrogen bonds with polar groups of the phospholipids [18]. As a matter of fact, several reports inform about different experimental log*D* values from the predicted log*P* values calculated exclusively for the neutral species of drugs [58,59].

Rocher et al. reported the percentage of the undissociated forms of i.e., CinA, *p*-CoA and FA in pulvinar cells of *Mimosa pudica* L. at pH 5.2, which amounted to 70.4%, 58.6%, and 52.7% of the total pool of these acids respectively [18]. Considering the fact that only lipophilic neutral forms are able to cross the plasma membrane by diffusion, this parameter bears important information on this family of carboxylic acids [60].

Regarding the importance of physicochemical properties of drugs, Lipinski developed Ro5 [18]. According to this rule, candidates for efficient drugs should be characterized by log*P* ≤ 5, molecular weight ≤ 500, number of hydrogen bond acceptors (O) ≤ 10, and number of hydrogen bond donors (OH, NH) ≤ 5. All phenolic acids examined herein meet the Lipinski's rule as demonstrated in Table 7. Thus, CinA, *p*-CoA and FA display favorable drug-like properties, which certainly encourage their further examination in the in vitro and in vivo studies.

Since phenolic compounds has been demonstrated as efficient cytostatic agents against various malignancies, it seems essential to evaluate how these compounds interact with lipid membranes, to get fuller insight into the transportation and anticancer mechanisms of these natural compounds. The affinity of polyphenols to the lipid bilayer is reflected in several electrochemical parameters. The first determinant is the adsorption on the membrane surface mediated by interactions of hydrophilic parts with the polar head groups of the lipids at the water–lipid interface. Second is the absorption dependent on the partitioning of the hydrophobic parts into the nonpolar core of the membrane [61,62]. The mechanism of action of polyphenols is determined by the presence of different substituents in their backbone structure and the pH value of their microenvironment. If the pH of the external medium is low (acidic), phenolic acids are then able to diffuse through the membrane due to their unchanged form [63,64]. The ELS data presented in Figures 2–4 clearly show that cinnamic acid and its derivatives do not affect the surface charge density of liposomal membranes at acidic pH. Therefore, it can be assumed that at acidic pH, investigated polyphenols were able to solubilize into the membrane and to permeate it. At neutral and basic pH, the CinA was unable to considerably modify the surface charge of the model membrane due to the lack of a hydroxyl group. Whereas more hydrophilic *p*-CoA and FA remained anchored at the bilayer surface without perturbing the lipidic structure but clearly affecting σ values. These findings are in agreement with literature, where the influence of structural characteristics of cinnamic acid and its hydroxyl derivatives on their interaction with model membranes was reported [51,65,66]. Similarly, the penetration of many other compounds e.g., flavonoids [7] or non-steroidal anti-inflammatory drugs [67] in bilayers, depends on the pH of the media.

Given the *p*KCOOH values (Table 7), at neutral and basic pH, the carboxyl group of CinA, *p*-CoA and FA is most likely negatively charged, and the negative charge of their molecules is probably oriented towards the positive pole of zwitterionic DOPC headgroup. ELS measurements performed in the presence of two concentrations of phenolic acids corroborate this hypothesis given that the surface charge of the membrane was altered, becoming increasingly negative with the addition of the examined polyphenols. The same tendency was observed for other negatively charged compounds [67]. The incorporation of cinnamic acid resulted in the slightest changes of σ, which again suggests interactions of CinA with the interior of the membrane. It is obvious that the effects of different phenolic

acids are correlated to their structural characteristics, thereby even the difference in one –OH group can be important, as well as the number of H-bonds they form. CinA can form three H-bonds, with one as a H-bond donor, and two as H-bond acceptors; its partition coefficient was the highest, and it was the most soluble of these phenolic acids in octanol (Table 7). In other words, CinA as a non-polar substance could enter the lipid bilayer easily. Our findings are in line with the literature, where the interaction of model membranes with phenolic acids presenting different structural changes in their molecular backbone is deciphered on the basis of the shift of lipid phase transition temperature [51,61,66,68]. These authors reported that for pH below p*K*COOH values, the protonation of the carboxylic group allows the substance to penetrate the lipid bilayer. Likewise, Castelli et al. employed multilamellar or unilamellar liposomes created from synthetic L-R-dimyristoylphosphatidylcholine to check whether cinnamic acid can dissolve into lipid membranes and penetrate them by migration from the aqueous phase [51]. This process continues until there is a constant molar fraction on the membrane surface, and then progressively inside the other internal bilayers. At the end of this process, the thermotropic behavior is close to that obtained by direct mixing of the biophenol with the lipid component during the liposomal preparation. The same researchers investigating PC liposomes containing *p*-CoA at two different pHs (4 and 7) reported no shift of the calorimetric peaks toward lower values, suggesting that the –OH group influences the ability of this compound to penetrate the membrane [51]. They did not exclude a surface interaction with the lipid layers and stated that the presence of different substituents in the backbone structure of biophenols might influence their incorporation. On the other hand, Ota et al. compared the thermograms obtained for the unilamelar large vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the presence of the *p*-CoA or FA, and noticed that the enthalpy value of the main transition of the phospholipids decreased by 1.46 ± 0.10 kcal/mol in the presence of *p*-CoA, and by 0.45 ± 0.17 kcal/mol with FA. This small but significant decrease in the enthalpy of transition implies that both acids intercalate into the acyl chain region of the bilayer [66].

Analyzing the influence of the polar headgroup of lipids on the penetration capacity of cinnamic acid and its derivatives, we expected that phenolic acids will be less able to penetrate the negatively charged headgroup of PS lipids as compared with DOPC. Surprisingly, we have not noticed such an effect. We speculate that perhaps the acids concentrations used here were not sufficient to cause significant alterations of the membrane surface charge, or it may also be the result of certain limitations of the ELS technique per se. Likewise, Fadel et al. [64] reported that rosmarinic acid, another compound belonging to phenolic acids, evoked a weaker effect in PS than PC membranes.

Furthermore, we analyzed the influence of pH on the size of the liposomes. Our findings showed that for neutral liposomes (DOPC), their diameter increased with increasing pH of the solution. It may suggest that in acidic pH, existing electrostatic repulsive forces dominate over hydrogen bonding affinities between neighboring lipid molecules [69]. This might be a tentative explanation of why we observed smaller sizes of DOPC liposomes. Contrarily, for the negatively charged liposomes (DOPC/PS 9:1, DOPC/PS 8:2, PS), the diameter decreased with increasing pH. As such, at acidic pH, the extent of protonation was higher than this observed in the case of DOPC liposomes. Also, an opposite dependence, the dominance of hydrogen bonding over electrostatic repulsions, was observed. This might be the reason why the negatively charged liposomes showed larger sizes than neutral ones (DOPC). Simultaneously, in alkaline and neutral pH, reduction of the diameter of negatively charged liposomes was observed most probably due to the repulsive forces between phospholipid molecules [70].

The EIS data presented in Figures 6–8 and collected in Tables 4–6 indicate that the addition of cinnamic acid and its hydroxy derivatives to the DOPC, DOPC/PS 9:1 or DOPC/PS 8:2 membranes caused an increase in their resistance, and thus reduced conductivity. An increase in the *R*<sup>m</sup> value implies that these phenolic acids exacerbated the ordering and decreased the dynamics of the phospholipid alkyl chains of spherical bilayers. This behavior can be explained in relation to the polarities of the examined molecules, indicating to what levels they can penetrate into the lipid bilayer. CinA, the least polar acid among the tested ones, had the greatest effect on the structure of the membrane lipids (i.e., stabilizing of the structure). *p*-CoA and FA are both more polar than CinA (see Figure 1), therefore, presenting a weaker effect on the membrane resistance. Based on our experimental approach, the following order of the stabilization effect was established: cinnamic acid > *p*-coumaric acid > ferulic acid. Our results are in line with previously reported data where the influence of the phenolic acids on structural properties of a model lipid membrane was investigated by differential scanning calorimetry [51,66,68], fluorescence spectroscopy [66,68], and electron paramagnetic resonance spectroscopy [68].

As opposed to the resistance, alterations in the capacitance seem to be less obvious in Nyquist plots, therefore, no remarkable differences between subsequent EIS measurements can be observed in Figures 6–8. It may be stated that the deposition of phenolic acids on the membrane was not accompanied by the appearance of any additional time constant, which was also reported not to happen in the case of protein adsorption [71]. Consequently, the *C*<sup>m</sup> values fitted from the equivalent circuit model were in alignment with the indications from the Nyquist (Tables 4–6), revealing increasing capacitance with increasing phenolic acids concentrations.

Together, the EIS data indicate that in comparison to untreated plain phospholipid bilayer, CinA-stimulated membranes showed a significant and systematic increase in bilayer resistance, which is dependent on increasing the concentration of phenolic acid. These changes were accompanied by moderate elevation of the bilayer capacitance, which can be attributed to a decrease in membrane thickness. This in turn, may indicate the ordering and stabilizing effect of CinA on the phospholipid alkyl chains of bilayers.

According to the EIS response shown in Figures 6–8 and data collected in Tables 4–6, it is evident that *p*-CoA and FA interact with the bilayer in a different way than CinA. Both of these compounds elicit significant changes in the lipid membrane capacitance, which indicate their adsorption at the bilayer interface. In contrast to CinA, changes in membrane resistance caused by increasing concentrations of *p*-CoA or FA were much smaller. These observations were consistent with the ELS data, suggesting that the CinA associates strongly and penetrates deeply into the lipid membrane. Conversely, *p*-CoA and FA may locate near the phospholipid headgroup, most probably via electrostatic interaction, without perturbing the lipidic structure.

Finally, if we compare the influence of the polar headgroup of lipids on the *R*<sup>m</sup> and *C*<sup>m</sup> values of membranes modified with CinA, p-CoA or FA, it seems that phenolic acids are less able to interact with the negatively charged headgroup of PS lipids as compared with DOPC. This result is not surprising, because the data were registered at pH equals 6.59 (pH of 155 mM/L NaCl electrolyte solution), in which all tested compounds bear a negative charge (carboxylate group) that may cause charge repulsion between the two carboxylate functions of the serine in PS. Notably, we failed to identify the differences in the interaction between phenolic acids and DOPC or PS bilayers using zeta potential analysis. This indicates that impedance measurement is a useful and effective technique worth utilizing in physicochemical studies.
