Influence of the Biological Environment on the Photophysics of Photosensitizers
The efficiency of a photosensitizer used in photodynamic therapy is mainly defined by the photophysical processes that take place after light excitation. It should be noted that the environment in biological systems has a significant influence on the photophysics. In order to investigate these effects, photophysical studies were carried out depending on the environment.
By: Christian Reichardt // Maria Wächtler // Benjamin Dietzek
Photodynamic therapy (PDT) is a modern and innovative method of treating cancer in which an active substance, the so-called photosensitizer (PS), is selectively activated by light irradiation at the site of action. Here, the PS acts as a “mediator” by absorbing the energy of the irradiated light and transmitting it to cell components or oxygen, whereby reactive species such as singlet oxygen are formed and controlled cell death is initiated. Due to the targeted light irradiation, PDT is a minimally invasive procedure that minimizes unwanted damage to healthy tissue.
Ruthenium-based transition metal complexes, characterized by high stability, excellent absorption in the visible spectral range, and a very long-lasting triplet state that enables efficient energy transfer, have proven to be useful PS in PDT. Numerous cell studies have shown the high phototoxicity of these complexes. However, the photo-induced mechanisms that occur after photoexcitation of the PS within a few microseconds have been only partially investigated. Previous photophysical studies show results in simple solvents such as water. This approach does not take into account the influence of the specific cellular environment and specific interaction between cell components (e.g., cell nucleus or mitochondria) and the PS.
In order to close this conceptual gap, the first step was to investigate the photo-induced processes of a specific PS using time-resolved spectroscopy, in particular transient absorption spectroscopy, simulating a realistic biological environment. For this purpose, the influence of the ionic strength of the aqueous medium as well as the addition of DNA solution on the photo-induced processes was examined. A ruthenium complex carrying a pyrene group and characterized by a very long-lasting excited state (a few µs) was used as PS.
The influence of the medium on the photophysical processes in the PS was investigated using simulated bodily fluid, which represents a realistic ionic strength in cells. Concentration-dependent experiments have shown that the photo-induced processes, which lead to the formation of the long-lasting excited state, are accelerated by increasing the ionic strength. Furthermore, an additional process at very high ionic strengths can be observed, which is most likely based on the formation of ion pairs.
The addition of DNA has a significant influence on the photophysics of the PS, since the addition of PS to the DNA results in interactions with the base pairs. The lifetime of the excited state, which in water or simulated bodily fluid is in the range of 15 µs, is increased many times over to 40 µs by the addition of DNA. This can be justified by the intercalation of the PS in the DNA. This embedding protects the PS from external influences (e.g., formation of water adducts), which reduces the number of possible deactivation processes and thus prolongs the lifetime.
One approach to improving the PS in PDT is the targeted incorporation of the active substances within certain cell components. This produces a locally higher concentration of PS at the point of action, which leads to a more targeted energy transfer and thus also higher phototoxicity. For this purpose, cooperation partners at the University of Ulm and the Max Planck Institute for Polymer Research in Mainz have produced a PS that is specifically incorporated into the mitochondria of the cells by binding a peptide. The modification of the PS by the peptide has the positive effect that the lifetime of the excited state is extended from 300 ns to more than 700 ns by the binding of the peptide.
The experiments described here show that the biological environment (here: ionic strength, presence of DNA) has a decisive influence on the photophysics of the PS. Therefore, detailed studies of the photo-induced processes in realistic environments are necessary to understand the effect-determining processes in detail and to increase the efficiency of the PS for PDT.
Funded by: FCI, DFG