Electrochemical Impedance Spectroscopy to Evaluate the Adsorption and Structural Changes of Liposome Dependent with Surface Charge
Authors: Fujino Yusuke , Nakamura Ryo , Masuda Kyosuke , Yamashita Ichiro , Shimizu Tomohiro , Shingubara Shoso , Ito Takeshi 
Published: December 2020

Liposome is well known as mimicking a cell, which is composed of lipid bilayer membrane. Liposome is spherical shape and formed by amphiphilic molecules, meaning that outer layer is composed of hydrophilic molecules and inner layer is composed of hydrophobic molecules. Evaluation of adsorption and rupture of liposome on a surface is very important to drug delivery system (DDS) or understand the capability of the cell.
Many studies reported the adsorption and interaction of lipid bilayer using highly sensitive sensing methods without labeling such as quartz crystal microbalance (QCM), plasmonic devices such as SPR, LSPR and SERS, and electrochemical impedance spectroscopy (EIS). We focus on EIS as an evaluation tool for adsorption and rupture of liposome, since EIS is highly sensitive to the surface condition of the electrode and electrochemical measurement is simple.
EIS is very sensitive to the surface condition of the electrode. We report here that surface charge is very important to evaluate the adsorption and interaction of liposome on the electrode surface, which are controlled by using self–assembled monolayer (SAM). So far as we know, there are little reports focusing on surface charge evaluate the adsorption and interaction of liposome on the electrode surface. Here, we report the effect of surface charge on electrochemical reactions due to SAMs which were coated on the Au electrode.
Liposome preparation was described below. Thin organic film was formed with 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). Then we obtained solution including DOPC which diameter was lower than 220 nm because the solution was filtered 20 times through the syringe filter (pore diameter was 220 nm). We used 1-Dodecanethiol (DDT), 11-Mercapto-1-undecanol (MUD or 11–HUT: 11–Hydroxy–1–undecanthiol) and 11-Amino-1-undecanthiol (11–AUT) for the SAM. These chemicals have same straight–chain alkane with thiol group to bind with Au surface, but head group is changed as CH3 for DDT meaning that the surface is neutral and hydrophobic, CH2OH for MUD meaning that the surface is charged negative and hydrophilic and CH2NH2 for 11–AUT meaning that the surface is charged positive and hydrophilic. Thus, we can discuss about the characteristics of Rct dependent with head group of SAM on the detection of liposome adsorption and rupture. As the results, most sensitive surface was obtained on use of 11–AUT in this study. In addition, we estimated the critical micelle concentration (CMC) of a surfactant, Triton–X 100, on the rupture process of liposome.
As an example of results, we show two type of hydrophilic surface, type A and B. Type A was coated with 11-Mercapto-1-undecanol, which surface had hydroxy group and was negatively charged. Type B was coated with 11-Amino-1-undecanol, which surface had amino group and was positively charged.
At first, we discussed about the results on type A. Fig. 1(Type A) shows Nyquist plots according to adsorption and rupture of liposome. After the adsorption of liposome, Rct decreased drastically. This characteristic was caused by surface charge after the liposome adsorption. As written above, the hydroxyl surface was charged negatively which repel the redox probe. After the liposome adsorption, surface charge changed to positive because the surface of DOPC was charged positively, which attracted the redox probes by charge polarity. After adding the surfactant, Rct increased and was almost same as that before the liposome adsorption. Next, we discussed about the results on type B. Fig. 1(Type B) shows Nyquist plots according to adsorption and rupture of liposome. After the adsorption of liposome, Rct increased drastically due to steric barrier of liposome. In this case, liposome adhered on the surface by hydrophilic interaction. Surface charge was not changed via liposome adsorption. After adding the surfactant, Rct decreased and was almost twice comparing with that before the liposome adsorption. This data show that ruptured liposome and/or surfactant adhered on the electrode surface. As discussed above, the amino surface was superior to detect adhesion and rupture of liposome comparing with other electrodes which coated with/without methyl and hydroxy SAM, when [Fe(CN)6]3-/4- was used as redox probes because of mixed surface conditions such as hydrophilicity and electric charge.

Figure 1