Dynamics of Cell Membrane Permeabilization by Saponins Using Terahertz Attenuated Total Reflection

Abstract

Understanding the relevant parameters of the formation of pores during permeabilization is very challenging for medical applications. Several components are involved: the arrival of the permeabilizing molecules to the membrane, the efficiency of formation of the pores and their specific dynamics, and the flux of molecules through the plasma membrane. Using attenuated total reflection in the terahertz domain, we studied the dynamics of Madine-Darby canine kidney cells after permeabilization by saponin molecules. We developed an analytical model taking into account saponin molecule diffusion, cell geometry, cytosol molecule diffusion, and pore dynamics. We also studied the effect of possible pore overlapping on the cell membrane, introducing a dimensionless quantity that is the ratio between overlapping and diffusive effects. Pores are found to be static within 1 h after their creation, hinting that the diffusion of the saponin molecules to the membrane is the limiting factor in our experiments.

Figure 1

THz-ATR device inserted in the THz-TDS setup. (A) The sample is located on top of a silicon support put in close contact with the top of the silicon prism. The support can be translated. Polarization is in the incident plane (p-polarization). (B) Close view of the ATR prism is shown. The cell layer lies in the evanescent field zone (in color gradient). To see this figure in color, go online.

Figure 2

Model of the pore formation mechanism on the cell membrane. The cell layer is put on top of the prism substrate. To see this figure in color, go online.

Figure 3

THz-ATR signal Δ(t) for MDCK cell layer after addition of saponins at T = 0 for experimental data (black dots) and best fit (red line). The blue vertical line shows addition of saponin. To see this figure in color, go online.

Figure 4

Model parameters φ (A) and 1/τ (B) vs. saponin mass concentration c₀. (A) The line is the best-fit proportional function. (B) The gray rectangle is a guide for the eye. The black line is the apparent 1/τ evolution considering overlapping of the pores. Inset shows the distribution of 1/τ for the whole concentrations and a Gaussian fit. For both, the number of experiments is shown in parentheses. To see this figure in color, go online.