Synthetic Strategy for Controllable Electrochemical Regulation of Metabolism in Living Cells Using Biocompatible Amphiphilic Redox-Active Polymers
Authors: Kaneko Masahiro , Ishikawa Masahito , Kato Souichiro , Hashimoto Kazuhito , Nakanishi Shuji 
Published: February 2020

In general, metabolism in living cells is closely related to the intracellular redox state. Focusing on the fact, a lot of studies examined the electrochemical regulation of cellular metabolism using lipophilic electron mediators with transmembrane ability. In these systems, electron mediators transport electrons between the intracellular redox active species and the extracellular redox active species or electrodes, across cell membranes. This process is called extracellular electron transfer (EET). Although conventional lipophilic mediators show cytotoxicity and are not suitable for long-term cultivation of living cells, we have recently developed a redox-active polymer as a biocompatible electron mediator (hereafter, the mediator is called PMF)[1]. PMF is an amphiphilic, transmembrane random copolymer polymerized by a free radical polymerization of hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC) and hydrophobic vinyl ferrocene (VFc). The MPC and the VFc unit plays a role as a biocompatible and a redox active unit, respectively. Using thus synthesized PMF, we have successfully achieved electrochemical regulations of microbial metabolism without any genetic manipulations[2]. In the present work, with the aim of expanding the potential of this method, we attempted to regulate EET efficiency by tuning the molecular structure of the PMF polymers.
It is known that the transmembrane ability is one of the rate determining factors in EET with conventional lipophilic electron mediators. Therefore, we assumed that cell membrane permeability could also influence the efficiency of the PMF-mediated EET. To verify this hypothesis, we added ethylene diamine tetra acetic acid (EDTA), which is known as a reagent to increase the cell-membrane permeability of gram-negative bacteria, during the measurement of the EET current from E. coli. Consequently, the EET current was increased by the addition of EDTA, and thus, the importance of the cell-membrane permeability was confirmed also for the case of the PMF-mediated EET. On the basis of the results, we decided to control the transmembrane ability of PMF for improving the EET efficiency. Theoretical calculations on amphiphilic copolymers have indicated that there is an suitable ratio between hydrophilic and hydrophobic units (R) for efficient cell-membrane permeability[3]. In addition, in the case of random copolymers including PMFs, cell-membrane permeability is expected to increase with decreasing molecular weight (Mw
).
Based on these assumption, three kinds of PMF polymers with different Mw and R values (in this case, the ratio of VFc in the PMF polymer) were synthesized in this study. To evaluate the effects of three PMF polymers on EET, EET current from E. coli and S. cerevisiae was measured. Here, E. coli and S. cerevisiae were used as model living cells as prokaryotic cells and eukaryotic cells, respectively. From both experiments, the tendency of the relationship between EET efficiency and properties of PMF was revealed to be nearly the same for these two species. Experimental results suggested that R has a more significant influence on EET efficiency. Furthermore, it was also suggested that the EET efficiency could be increased with decreasing Mw at similar R values, as expected.
Next, we tried to investigate the relationship between EET efficiency and the metabolisms by evaluating the activity of anaerobic glycolysis of S. cerevisiae as a model system. It is reported that lipophilic electron mediators with the redox potential lower than redox active species in electron transport chain (ETC) change the metabolic pathway of S. cerevisiae from the anaerobic glycolysis to anaerobic respiration by intercepting intracellular electrons from the ETC[4]. To evaluate the change of the metabolic pathway, we measured ethanol concentrations and the biomass in cell culture cultivated in the presence or the absence of the oxidized form of PMF. It was anticipated that the ethanol production becomes lower and the biomass becomes higher. In fact, the metabolic pathway was revealed to change in the presence of the oxidized form of PMF. Reasonably, the degree of the metabolism change was higher when the PMF with higher EET efficiency was used. These results indicated that the properties of the PMFs affects the efficiency of electrochemical regulation of cellular metabolism.

Reference

[1]Nishio et al, Chemphyschem, 2013, 14, 2159–2163.
[2]Nishio et al, Environ. Sci. Technol. Lett., 2014, 1, 40–43.
[3]Werner et al, Biomacromolecules, 2015, 16, 125–135.
[4]Zhao et al, Anal. Chim. Acta, 2007, 597, 67–74.

Figure 1