Terahertz Spectroscopy Sheds Light on Real-Time Exchange Kinetics Occurring through Plasma Membrane during Photodynamic Therapy Treatment

Abstract

Methods to follow in real time complex processes occurring along living cell membranes such as cell permeabilization are rare. Here, the terahertz spectroscopy reveals early events in plasma membrane alteration generated during photodynamic therapy (PDT) protocol, events which are not observable in any other conventional biological techniques performed in parallel as comparison. Photodynamic process is examined in Madin-Darby canine kidney cells using Pheophorbide (Pheo) photosensitizer alone or alternatively encapsulated in poly(ethylene oxide)-block-poly(ε-caprolactone) micelles for drug delivery purpose. Terahertz spectroscopy (THz) reveals that plasma membrane permeabilization starts simultaneously with illumination and is stronger when photosensitizer is encapsulated. In parallel, the exchange of biological species is assessed. Over several hours, this conventional approach demonstrates significant differences between free and encapsulated Pheo, the latter leading to high penetration of propidium iodide, Na⁺ and Ca²⁺ ions, and a high level of leakage of K⁺ , ATP, and lactate dehydrogenase. THz spectroscopy provides, in a single measurement, the relative number of defects per membrane surface created after PDT, which is not achieved by any other method, providing early, sensitive real-time information. THz spectroscopy is therefore a promising technique and can be applied to any biological topic requiring the examination of short-term plasma membrane permeabilization.

Keywords: permeability; photodynamic therapy PDT; plasma membrane; polymers; self-assembly.

Figure 1

Cell integrity was drastically and rapidly affected by PDT with encapsulated photosensitizer. General aspect of MDCK1 cells was observed by scanning electron microscopy at different short time points after exposure to light irradiation. Pictures are representative of what was observed in the total cell population in two independent experiments. c indicates cells. Arrows indicate unroofed cells. Scale bar = 20 µm.

Figure 2

Pheo encapsulation enhances its effects on molecules exchanges across the plasma membrane after PDT treatment. Quantification of molecules of different molecular weights in the extracellular medium at different time points after MDCK1 cells PDT treatment with free or encapsulated photosensitizer. A) ATP release quantified by luminescence. n = 6. B) Lactate dehydrogenase enzyme (LDH) activity quantified by absorbance. n = 6. C) Ionic movements 30 min after PDT treatment quantified by ICP‐OES and ionic chromatography. n = 6. Data are represented as mean ± SEM and analyzed by one‐way ANOVA followed by Dunnett's multiple comparisons test, which compare all conditions with the control condition. ● = control,  = empty micelles,   = Pheo alone,   = Pheo‐micelles.

Figure 3

Pheo encapsulation accentuated its effects on loss of plasma membrane integrity. A) Real‐time monitoring of plasma membrane integrity through propidium iodide penetration, quantified by fluorescence videomicroscopy over time after PDT treatment with increasing concentrations of free or encapsulated photosensitizer. B) Focus on 4 h time point after light irradiation. C) Focus on 15 h time point after light irradiation. D) Amplitude parameter Δ extracted from propidium iodide penetration observed by videomicroscopy. E) Decay time T for fluorescence extracted from propidium iodide penetration observed by videomicroscopy. Data are represented as mean ± standard error of the mean and analyzed using unpaired t‐test. n = 4.  = Pheo alone,  = Pheo‐micelles.

Figure 4

Dual‐modulation differential ATR sensor setup. A) 3D view. B) Detailed view. The THz beam from the QCL source is chopped at two different frequencies x5 and x6 by the two rows of chopper slots. Half of the beam is modulated at x6 and is reflected by the sample, while the other is modulated at x5 and used as a reference. The two halves are recombined and sent to the same detector. Two lock‐in amplifiers are used to detect the sample and reference signals. The chopper controller provides the modulation signals for the optical chopper and lock‐in amplifiers.

Figure 5

Dynamics of plasma membrane permeabilization following free or encapsulated PDT, analyzed by THz‐ATR. A) Summary of illuminated conditions. Mean values (number of replicates is indicated in brackets) are represented. Concentration of Pheophorbide is 1.65 µm. B) Amplitude parameter Δ for all conditions. C) Amplitude parameter Δ for illuminated condition in presence of Pheo alone and Pheo‐micelles, for different concentrations (in µm). D) Delay time T for illuminated condition in presence of Pheo alone and encapsulated Pheo in micelles, for different concentrations (in µm). The right‐hand axis gives the relative number of defects per membrane surface Σ/S. Values at 0.0825 and 0.165 µm are not accessible for Pheo alone since the amplitude of the signals is close to 0. ■: pheo alone condition; ▼: pheo encapsulated in micelles condition; ▲: empty micelles condition; and ●: control condition.