*3.3. The E*ff*ect of Cinnamic Acid and Its Derivatives on Capacitance and Resistance of Spherical Bilayers*

− − − Electrochemical impedance spectroscopy was utilized to quantify the effect of the examined phenolic acids on the membrane resistance (reciprocal of conductance) and capacitance. This method seems to be non-destructive and presents high sensitivity to feasible drug-induced modulation of bilayer thickness or packing [48]. As such, EIS was used over a wide range of frequencies from 10−<sup>1</sup> Hz to 10<sup>4</sup> Hz under small amplitude. The 155 mM/L NaCl electrolyte solution was used to register impedance spectra.

Two different phospholipids i.e., DOPC and PS, were used to prepare one- or two-component bilayers. Unfortunately, bilayers composed only from PS did not have the ability to form sufficiently stable membranes for the impedance measurements. CinA, *p*-CoA or FA was introduced to the phospholipid model membrane solution at concentrations of 1 and 5 mM/L. In order to extract the *R*<sup>m</sup> and *C*<sup>m</sup> values from the impedance spectra, the EIS data were fitted to the equivalent circuit model shown in Scheme 1 presented in section "Materials and Methods". In the following analyses, the values of the impedance parameters referred to the bilayer surface area unit. At least six spherical lipid bilayers were tested for consistency. For quantitative data, average values with standard deviations are reported.

Figure 6 displays the impedance response in the form of Nyquist plots of different spherical bilayers, with varied concentrations of CinA. Notably, the plot registered for each bilayer contains a capacitive arc across the entire frequency range. The diameter of the arc corresponds to the membrane resistance. The incorporation of the CinA into the DOPC, DOPC/PS 9:1 or DOPC/PS 8:2 membrane resulted in an increase in the *R*<sup>m</sup> values (compared to the values obtained for bilayers with the -2















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DOPC/PS 8:2

DOPC/PS 8:2

DOPC/PS 8:2

DOPC/PS 8:2 + 1 mM p-CoA

DOPC/PS 8:2 + 1 mM p-CoA

DOPC/PS 8:2 + 1 mM p-CoA

**pH**

**pH**

**pH**

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2




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Ω

same lipid composition but without phenolic acid), confirming that this compound is active at the tested concentrations.

Ω

μ

Ω

■ ■ ■ **Figure 6.** Representative impedance spectra for (**a**) DOPC, (**b**) DOPC/PS 9:1, and (**c**) DOPC/PS 8:2 osomal membranes as a function of ( spherical bilayers as a function of (■) 0 ■ ■ ■) 0, ( , (■) 1, and (■ ■ ■) 1, and ( ) 1, and (■) ) 5 mM/L of cinnamic acid concentration. Experimental data points are indicated with markers; model fits are shown with lines.

0 2 4 6 810 **pH** DOPC DOPC + 1 mM p-CoA DOPC + 5 mM p-CoA -4 -2 0 2 0 2 4 6 810 *б* **[10-2 C/m2 ] pH** DOPC/PS 9:1 DOPC/PS 9:1 + 1 mM p-CoA DOPC/PS 9:1 + 5 mM p-CoA 0 2 4 6 810 **pH** DOPC DOPC + 1 mM p-CoA DOPC + 5 mM p-CoA -4 -2 0 2 0 2 4 6 810 *б* **[10-2 C/m2 ] pH** DOPC/PS 9:1 DOPC/PS 9:1 + 1 mM p-CoA DOPC/PS 9:1 + 5 mM p-CoA 0 2 4 6 810 **pH** DOPC DOPC + 1 mM p-CoA DOPC + 5 mM p-CoA -4 -2 0 2 0 2 4 6 810 *б* **[10-2 C/m2 ] pH** DOPC/PS 9:1 DOPC/PS 9:1 + 1 mM p-CoA DOPC/PS 9:1 + 5 mM p-CoA Table 4 provides the values of the electrochemical elements obtained by fitting the electrochemical impedance spectra. The typical value of the electrolyte resistance was equal to (5.57 ± 0.20) × 10<sup>3</sup> Ω and was considered irrelevant to the bilayers characteristics. Therefore, this parameter was omitted in the curve fitting procedure. The *R*<sup>m</sup> value of the DOPC bilayer was evaluated prior to the addition of the phenolic acid and found to be (1.472 ± 0.07) × 10<sup>6</sup> Ω cm<sup>2</sup> , which is consistent with the reported values for other PC known to typically range between 10<sup>4</sup> and 10<sup>7</sup> Ω cm<sup>2</sup> [30,31]. The obtained *C*<sup>m</sup> value for plain DOPC membrane was (0.617 ± 0.03) µF/cm<sup>2</sup> , which is in line with the capacitance values of PC bilayers calculated based on the chronopotentiometric [27], chronoamperometric [49] or impedance [45] method. As presented in Table 4, the increase in membrane capacitance was also noticed after the addition of CinA to the spherical bilayers.


0 2 4 6 810

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0 2 4 6 810

PS

PS

PS

**pH**

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PS + 1 mM p-CoA PS + 5 mM p-CoA

PS + 1 mM p-CoA PS + 5 mM p-CoA










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**Table 4.** Effect of cinnamic acid on the resistance and capacitance of lipid bilayer.

**Ω <sup>−</sup> μ**

♦ ♦ ♦ **Figure 7.** Representative impedance spectra for (**a**) DOPC, (**b**) DOPC/PS 9:1, and (**c**) DOPC/PS 8:2 spherical bilayers as a function of ( of (♦) 0 ♦ ♦ ♦) 0, ( ) 0, (♦) ♦ ♦ ♦) 1, and ( 1, and (♦) a ) 5 mM/L of *p*-coumaric acid concentration. Experimental data points are indicated with markers; model fits are shown with lines.

Table 5 summarizes the results concerning the influence of the membrane composition on the capacitance and resistance values. Upon introducing *p*-CoA, an increase in both parameters was detected compared to the plain membranes.

*σ*

*σ*

*σ*

**pH**

**pH**

**pH**

**pH**

**pH**

**pH**

DOPC

DOPC

DOPC

DOPC + 1 mM FA DOPC + 5 mM FA

DOPC + 1 mM FA DOPC + 5 mM FA

DOPC + 1 mM FA DOPC + 5 mM FA

*σ*

*σ*

*σ*










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0 2 4 6 810

0 2 4 6 810

0 2 4 6 810

0 2 4 6 810

0 2 4 6 810

DOPC/PS 8:2

DOPC/PS 8:2

DOPC/PS 8:2

DOPC/PS 8:2 + 1 mM FA DOPC/PS 8:2 + 5 mM FA

DOPC/PS 8:2 + 1 mM FA DOPC/PS 8:2 + 5 mM FA

DOPC/PS 8:2 + 1 mM FA DOPC/PS 8:2 + 5 mM FA

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DOPC/PS 9:1

DOPC/PS 9:1

DOPC/PS 9:1

DOPC/PS 9:1 + 1 mM FA DOPC/PS 9:1 + 5 mM FA

DOPC/PS 9:1 + 1 mM FA DOPC/PS 9:1 + 5 mM FA

DOPC/PS 9:1 + 1 mM FA DOPC/PS 9:1 + 5 mM FA

**pH**

**pH**

**pH**

**pH**

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PS + 1 mM FA PS + 5 mM FA

PS + 1 mM FA PS + 5 mM FA

PS + 1 mM FA PS + 5 mM FA

PS

PS

PS













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**Table 5.** Effect of *p*-coumaric acid on the resistance and capacitance of lipid bilayer.

From the subsequent EIS data presented in Figure 8 and Table 6, it is evident that both the bilayer resistance and capacitance increase in the presence of FA. ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦

▲ ▲ ▲ **Figure 8.** Representative impedance spectra for (**a**) DOPC, (**b**) DOPC/PS 9:1, and (**c**) DOPC/PS 8:2 spherical bilayers a function of ( function of (▲) 0, ▲ ▲ ) 0, ( ▲) 0, (▲) 1, an ▲ ) 1, and ( ▲ ▲ d (▲) 5 mM/L ) 5 mM/L of ferulic acid concentration. Experimental data points are indicated with markers; model fits are shown with lines.

∼ ∼

∼ ∼

∼ ∼


**Table 6.** Effect of ferulic acid on the resistance and capacitance of lipid bilayer.

Taking into account the data gathered in Figures 6–8 and in Tables 4–6 and by comparing membranes with the same phospholipid composition, it may be noted that the smallest changes in the capacitance and the biggest changes in the resistance values were observed in the presence of the most hydrophobic of all tested compounds—cinnamic acid. Conversely, the largest alterations in the capacitance and the lowest in the resistance were noticed in the presence of the most hydrophilic one, ferulic acid.
