Terahertz ATR sheds light on real-time exchange kinetics occurring through plasma membrane during photodynamic therapy

THz ATR spectroscopy provides, in a single measurement, the relative number of defects per membrane surface created by oxidative stress generated during photodynamic therapy (PDT), offering early, sensitive real-time information. THz spectroscopy is therefore a complementary technique to established (biological) assays and can be applied to any topic requiring the real-time examination of short-term plasma membrane permeabilization.


I. INTRODUCTION
ETHODS to follow in real time complex processes occurring along living cell membranes such as cell permeabilization are rare.Here, we show that terahertz spectroscopy reveals early events in plasma membrane alteration generated during a photodynamic therapy protocol, events which are not observable in any other conventional biological techniques performed in parallel as comparison [1].The photodynamic process was examined in MDCK1 cells using Pheophorbide a (Pheo) photosensitizer alone or alternatively encapsulated in poly(ethylene oxide)-blockpoly(-caprolactone) micelles for drug delivery purpose.Terahertz spectroscopy revealed that plasma membrane permeabilization started simultaneously with illumination and was stronger when photosensitizer was encapsulated.In parallel, the exchange of biological species was assessed.Over several hours, this conventional approach demonstrated significant differences between free and encapsulated Pheo, the latter leading to high penetration of propidium iodide, Na + and Ca 2+ ions, and a high level of leakage of K + , ATP and LDH.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 complementary technique to established assays and can be applied to any biological topic requiring the examination of plasma membrane permeabilization.

II. METHODS
Terahertz ATR uses the evanescent wave at the back of a prism under total internal reflection, which is coupled to the sample under study (Fig. 1).When an absorbing layer such as cells is placed on top of the prism, it couples with the evanescent field and reduces the amount of reflected energy.The measurement of the reflected beam is then directly related to the dielectric constant of the medium on top of the prism.More specifically, we consider a thin layer of cells and the surrounding solution in this inhomogeneous evanescent wave.Provided that the thickness of the cell layer matches the penetration depth of the evanescent wave, the reflected terahertz wave is modified by the terahertz dielectric properties of the cell layer in contact with the top of the prism.The stronger coupling is obtained for a penetration depth equal to the cell layer thickness.The resulting reflected terahertz signal is then correlated with the cytosol content [2][3][4], in real time without any marker or specific sample preparation.In our experiments, we used a differential setup based on a QCL source to obtain stable and precise measurements [5].
PDT is used clinically to treat various pathologies in dermatology, ophthalmology or oncology [6].It is based on the in-situ formation of Reactive Oxygen Species (ROS).These are formed by illumination of a photosensitizer, present at the site to be treated, which, upon excitation, exchanges its energy with the surrounding oxygen molecules.
MDCK cells are an immortal cell line derived from canine kidney.They are widely used as a model cell line for studies of epithelial polarization and transport, mechanisms of infection, regulation of tight junctions, etc.These cells form a typical cuboidal monolayer epithelium when grown to confluency, where they have a height of 10 to 12µm.

III. RESULTS
Standard biological laboratory experiments and physical measurements by THz spectroscopy were carried out in parallel to ensure the complementarity of the data collected, following the treatment of cancer cells by photodynamic therapy [1].The results shown in Fig. ~2 indicate a real added value of THz spectroscopy compared to biological reference approaches.In particular THz measurements exhibit much better sensitivity.For instance, dose effects for concentrations below 0.33µM could not be detected using standard biological methods.(Fig. ~2C and D).Indeed, this new methodology provides both a quantitative estimate of the number of membrane surface defects created by oxidative stress during photodynamic therapy, but also more efficient measurements than biological approaches conducted in parallel.It also allows to obtain early information on the development of cellular responses, i.e. from the moment of light irradiation, as well as a real-time monitoring of the intracellular water content, on time scales of several hours.THz spectroscopy is thus a promising technique complementary to established classical biological studies and techniques, which can be applied to any subject requiring realtime examination of the modulation of cell water content.In particular This study validates the original and innovative application of THz spectroscopy, a physical technique that is applicable to biological and medical issues.Eventually, the technological development of terahertz radiation sources will allow to determine with precision the nature of the chemical elements involved in the modulations of intracellular water, following external stimuli such as PDT.Authorized licensed use limited to the terms of the applicable license agreement with IEEE.Restrictions apply.

Fig. 2 .
Fig. 2. A. Summary of illuminated conditions.Mean values (number of replicates in brackets) are represented.Concentration of Pheophorbide is 1.65 µM.B. Amplitude parameter  for all conditions.C. Amplitude parameter  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.