In an article published in the Journal of the American Chemical Society (JACS), researchers led by Professor Mauricio S. Baptista of the Instituto de Química at Universidade de São Paulo (USP) and a member of the RIDC Redoxoma showed that photosensitizer (PS) degradation caused by contact-dependent reactions is a necessary step to cause irreversible damage to biological membranes. Therefore, in medical applications of photodynamic therapy, contact-dependent reactions can damage biological targets much more accurately than those in which diffusive species such as singlet oxygen and other reactive oxygen species are formed. The results can impact the design of more efficient photosensitizers.

“For decades researchers have focused on the same mechanism of action of photodynamic therapy, which is singlet oxygen generation. It is a paradigm. We now propose that contact-dependent reactions must be considered for the development of more efficient photosensitizers. Our work also breaks another paradigm in this area, according to which PS photobleaching causes loss of efficiency because they no longer generate singlet oxygen. Our results show that photosensitizers photobleaching induces membrane leakage, - and for that, the PSs have to be attached to the membrane,” said the researcher.

Photodynamic therapy, or PDT, involves chemicals known as photosensitizers, which are activated by visible light to produce oxidizing species capable of killing cancer cells and bacteria. This low invasive and low toxic therapeutic modality has been studied and employed in different branches of medicine, replacing or complementing conventional therapies.

The great advantage of photodynamic therapy in the treatment of tumors is to preserve the original tissue. But, according to Baptista, one of the technical problems is to deliver light efficiently at the required depth. Therefore the importance of developing photosensitizers that work with less light.

As antimicrobial therapy, the perspectives of PDT are also interesting. “I think using photodynamic therapy as an antimicrobial will make a difference considering the emergence of superbugs resistant to antibiotics because nothing resists to this kind of damage,” the researcher said.”

Hundreds of types of molecules have been tested and function well as photosensitizers. “We propose to investigate other important factors for the efficiency of these molecules besides the generation of excited state oxidizing species.””

Photobleaching

Several processes that require light absorption and the subsequent photoinduced action of photosensitizers, such as photosynthesis, for example, cause the photosensitizers photobleaching or photodegradation. Photobleaching causes loss light absorption capacity, i.e. photosensitizers no longer function. In medical applications, photostability is considered an essential feature for photosensitizers.

However, according to the researchers, recent evidence points to the key role of contact-dependent reactions, which often cause photosensitizers photobleaching. It calls into question the paradigm of photostability versus photosensitizer efficiency in medical applications.

In this study, the researchers produced a series of magnesium-porphyrazine complexes (MgPzs) capable of generating the same amount of singlet oxygen but with different redox properties. Porphyrazines are pigments analogous to porphyrins, but with greater redox activity. They then investigated the photobleaching mechanism of these complexes in solution and found that it is independent of singlet oxygen, occurring mainly via electron abstraction of surrounding electron-rich molecules (solvents or lipids). In the process, the complexes are reduced to a radical intermediate.

Photoinduced cell death by a photosensitizer is associated with membrane damage. To correlate photobleaching with membrane permeabilization, the researchers compared two photosensitizers (CF₃Pz and FPz) with similar photophysical properties but with distinct photobleaching efficiencies. They used two membrane models, the liposomes, which are small spherical phospholipid vesicles, and the giant unilamellar vesicle (GUV), which can be observed under a microscope, and found that the higher the rate of PS photobleaching the faster the leakage induced in the membranes. The photobleaching occurs through an electron abstraction from the lipid double bond, which causes irreversible membrane damage.

“This work is a continuation of an earlier study also published in the JACS in which we compared two compounds, one bonded to the membrane and one not bonded, and we saw that the bonded compound caused more membrane damage, through the generation of aldehydes, and that it was degraded. Now we have made a system in which both compounds are bonded to the membrane and the only parameter that matters is their redox property. One of them is a more active oxidant and it is the one that undergoes the greatest photodegradation and permeabilizes the membrane more ”, explains Baptista.

This study was conducted as a postdoctoral project by Thiago T. Tasso, currently a professor at the Universidade Federal de Minas Gerais, first author and corresponding co-author of the article, with the collaboration of researchers from the Universidade Federal do ABC and the Departamento de Química Fundamental of the Instituto de Química at Universidade de São Paulo (USP).

The article Photobleaching Efficiency Parallels the Enhancement of Membrane Damage for Porphyrazine Photosensitizers, by Thiago T. Tasso, Jan C. Schlothauer, Helena C. Junqueira, Tiago A. Matias, Koiti Araki, Erica Liandra-Salvador, Felipe CT Antonio, Paula Homem-de-Mello and Mauricio S. Baptista, can be accessed at https://pubs.acs.org/doi/10.1021/jacs.9b05991