*3.1. The E*ff*ect of Cinnamic Acid and Its Derivatives on Surface Charge Densities of Liposomal Membranes*

To get insight into the surface charge density-modulatory properties of CinA, *p*-CoA and FA, the ELS technique has been engaged. The measurements were performed as a function of H<sup>+</sup> concentration. In order to receive the values of pH-dependent electrophoretic mobility, liposomes were suspended in 155 mM/L NaCl solution, which was further titrated to the adequate pH with concentrated NaOH or HCl. Surface charge density values were calculated on the basis of electrophoretic mobility in accordance to Equation (1) provided in section "Materials and Methods". Representative plots from at least three independent experiments are shown.

Surface charge density dependence on pH of plain and CinA-modified DOPC, DOPC/PS 9:1, DOPC/PS 8:2 and PS liposomal membrane occurred to be similarly shaped (Figure 2). An increase in positive surface charge density together with the decrease in pH values was noticed, however, only to a certain point. Conversely, along with the increasing pH, the negative charge of the membranes incremented until the plateau was reached. The visible decrease in the surface charge of PC-containing membranes at pH ~ 8.0 was possibly caused by the destruction of the membrane structure at such a high pH, which is in line with earlier reported data [7]. Our results demonstrated that in low pH, any visible alterations in values of the surface charge density were detected for all tested liposomal membranes treated with CinA. Nevertheless, at high pH values, the presence of CinA resulted in visible changes in surface charge densities of the aforementioned membranes (the negative charge of the membranes increased) compared to data obtained for plain phospholipid liposomal membranes. Those changes were apparently dose-dependent.

*σ* The liposomes treated with *p*-coumaric acid were examined analogously (Figure 3). It was found that in acidic solution, the presence of *p*-CoA did not affect the σ values. However, in alkaline solutions, an increase in the negative charge of *p*-CoA-treated membranes in comparison to plain phospholipid liposomal membranes was observed.

DOPC

DOPC

DOPC

DOPC + 1 mM CinA

DOPC + 1 mM CinA

DOPC + 1 mM CinA










*σ*

*σ*






*б*

**[10-2 C/m2**

*б***C/m**

**-2 ]**

**]**




0

0

2

2


*б*

*б*

*б*

**[10-2 C/m2**

**[10-2 C/m**

**-2 C/m2]**

**]**

0

0 2 4 6 810

4 6

0 2 4 6 810

0 2 4

DOPC/PS 8:2

∼∼-2-1

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

DOPC/PS 8:28:2 + mM FADOPC/PS 8:2 + 5 mM FA

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

1

2






*б*

*б*

*б*

**[10-2 C/m2**

**[10-2 C/m2**

**[10-2 C/m2**

**]**

**]**

**]**




0

0

0

2

2

2



*б*


*б*

*б*

**[10-2 C/m2**

0

**[10-2 C/m2**

**[10-2 C/m2**

**]**

**]**

1

**]**

0

0

1

1

2

2

2







*δ* **[10-2 C/m2**

*δ* **[10-2 C/m2**

*δ* **[10-2 C/m2**

**]**

**]**

**]**

0

0

0

2

2

2




*δ* **[10-2 C/m2**

*δ* **[10-2 C/m2**

*δ* **[10-2 C/m2**

**]**

**]**

**]**

0

0

0

1

1

1

2

2

2

2

2

2

DOPC/PS 9:1

DOPC/PS 9:1

DOPC/PS 9:1

DOPC/PS 9:1 + 1 mM CinA

DOPC/PS 9:1 + 1 mM CinA

DOPC/PS 9:1 + 1 mM CinA

■ ■ ■ **Figure 2.** pH dependence of surface charge density of (**a**) DOPC, (**b**) DOPC/PS 9:1, (**c**) DOPC/PS 8:2, and (**d**) PS liposomal membranes as a function of ( osomal membranes as a function of (■) 0 ■ ■ ■ ) 0, ( , (■) 1, and (■ ) 5 mM/L of cinnamic acid concentration. The results represent mean values from three independent experiments run in triplicate. ■ ■ ■


**pH**

**pH**

**pH**

**pH**

mM FA

▲▲▲

DOPC

DOPC DOPC DOPC

DOPC + 1 mM FA DOPC + 5 mM FA *σ*







*б*

**[10-2 C/m2**

*б***[10C/m**

**]**

**]**


0

2


*б*

**[10-2C/m2**

*б***[10C/m]**

**]**

**]**


0

0 2 4 6 810


0 2 4 6 810

2 4

0 2 4 6 810

DOPC/PS 9:1

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

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

9:1 9:1

**pH**

**pH**

PS + 1 mM FA PS + 5 mM FA

**pH**

PS

2

▲ ▲ ▲

∼ ∼










*б*


*б*

*б*

**[10-2 C/m2**

**]**

**[10-2 C/m2**


**[10-2 C/m2**

**]**

0

2

**]**



0

0

2

2



*б*

*б*

*б*

**[10-2 C/m2**

**]**

**[10-2 C/m2**

**[10-2 C/m2**

**]**

1

2

**]**

0

0

0

1

1

2

2

The surface charge densities of the DOPC, DOPC/PS 9:1, DOPC/PS 8:2, and PS liposomal membrane, plain and modified with ferulic acid, are shown as a function of pH in Figure 4. Again, the presence of the phenolic acid did not affect the σ values at low pH, but caused an increase in the negative σ values at higher pH ranges. *σ σ σ σ σ σ σ σ*

♦ ♦ ♦

♦ ♦ ♦

♦ ♦ ♦

♦ ♦ ♦

▲ ▲ ▲ **Figure 4.** pH dependence of surface charge density of (**a**) DOPC, (**b**) DOPC/PS 9:1, (**c**) DOPC/PS 8:2, and (**d**) PS liposomal membranes as a function of ( function of (▲) 0, ▲ ▲ ) 0, ( ▲) 0, (▲) 1, an ▲ ) 1, and ( ▲ ▲ d (▲) 5 mM/L ) 5 mM/L of ferulic acid concentration. The results represent mean values from three independent experiments run in triplicate.

Considering the ELS data presented in Figures 2–4 and comparing membranes with the same phospholipid composition, it can be observed that the smallest changes in the surface charge density values were obtained in the presence of the most hydrophobic of all, cinnamic acid, whereas the most prominent alterations were noticed in the presence of the most hydrophilic compound, ferulic acid.

∼ ∼ ∼ ∼ ∼ ∼ ∼ ∼ Finally, the results collected in Figures 2–4 illustrate that the presence of CinA, *p-*CoA or FA did not change the value of the isoelectric point of all analyzed phospholipid membranes. Since the isoelectric point of the membrane is one of the most important parameters describing its variable-charge surfaces, it is important to point out that it has shifted towards lower pH values for DOPC as compared to PS (decrease from pH ∼3.5 to pH ∼1.7, respectively). This is coherent with other investigations demonstrating that the isoelectric point of PC lipids fits into the pH values ranging between 3.2 and 4.0 [41,46,47], while reaching 1.4–1.7 values for PS lipids [41,46].
