What do spoiled food, skin cancer and photodynamic therapy have in common? If you thought of oxidizing lipid membranes, you got it right. All cells are surrounded by a membrane, formed by a lipid bilayer with associated proteins, which has among its functions to define the limits of the cell, separating the interior from the outside, and delimiting internal compartments. The phospholipids form the structural base of this bilayer and tend to undergo oxidation, damaging the membranes, which can make them permeable and cause cell death. When these oxidations are induced by light, which increases the speed of reactions, they are called photooxidations.

The general mechanisms of lipid oxidation have been known for a long time, but for photooxidations it was still necessary to show the chemical steps by which photosensitizers and light permeabilize lipid membranes, causing biological damage, as well as to characterize the products of these oxidations.

This was the focus of the study led by the team of Maurício da Silva Baptista, professor at the Instituto de Química of Universidade de São Paulo (USP) and a member of the Center for Research of Redox Processes in Biomedicine (RIDC Redoxoma) and published in July in the Journal of the American Chemical Society (JACS). The researchers demonstrated that permeabilization of the membrane is linked to the presence of lipid aldehydes formed in processes that depend on direct contact between photosensitizers and lipids. Thus, the study explains at the molecular level why membrane-bound photosensitizers destroy cells more effectively.

"Our work can provide mechanistic guidelines for new developments in photomedicine and photoprotection. The knowledge generated by this study, which is centered on basic science, indicates several application possibilities. For example, if we want to have a more effective photosensitizer, we have to find new ways to regenerate it. As a strategy for developing more efficient sunscreens, we need to create a way to prevent aldehydes from building up", said Baptista.

The study was carried out as a doctoral project of Isabel Bacellar, the paper’s first author, and counted with the collaboration of RIDC Redoxoma researchers Paolo Di Mascio and Sayuri Miyamoto. The work also included the groups of Professors Ronei Miotto and Rodrigo Maghdissian Cordeiro (Universidade Federal do ABC), Professor Gonzalo Cosa (McGill University, Canada) and Professor Mark Wainwright (Liverpool John Moores University, UK).

Photo Induced Oxidation and Aldehydes

Photosensitized oxidations are reactions elicited by the interaction of light with a photosensitizer molecule in the presence of oxygen. They have well-known harmful biological effects, such as skin aging and cancer, caused by exposure to sunlight. On the other hand, as Baptista explains, these same reactions can be used for disease treatment, as in the case of photodynamic therapy (PDT), in which they trigger the oxidation of biomolecules and thereby eliminate cancerous or pathogenic cells.

Singlet oxygen is generally considered the most important oxidant involved in photosensitized lipid oxidations. In these processes, the photosensitizer in the excited triplet state can interact with ground state oxygen molecules generating singlet oxygen, which is a more reactive form of oxygen, through an energy transfer process. This singlet oxygen reacts with unsaturated lipids, forming hydroperoxides, by addition to the double bonds of the biomolecules. Lipid membranes are important targets of photosensitized oxidations, undergoing various transformations, many of which are attributed to the action of lipid hydroperoxides.

However, according to Baptista, only the singlet oxygen action, which is a diffusing molecule, does not explain the known fact that photosensitizers that bind to membranes are more effective in making them permeable and therefore in destroying cells.

To investigate the reactions and compounds that lead to photoinduced membrane permeabilization, the researchers used two phenothiazinium photosensitizers, methylene blue (MB) and DO15. Both compounds have similar photophysical properties and are chemically similar as they are based on the same chromophore. The difference between them is that methylene blue is more hydrophilic and DO15 is more hydrophobic, hence their interaction with the membranes and the damage they cause are different. As membrane models, they used liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a lipid containing a monounsaturated fatty acid, as well as liposomes made with lipids containing 1-palmitoyl- 2-arachidonoyl-sn-glycero-3-phosphocholine, a polyunsaturated fatty acid.

To compare the lipid oxidation products formed by singlet oxygen with those formed by contact-dependent reactions, they designed experiments in which both photosensitizers delivered nearly the same amount of singlet oxygen to the membranes. The difference was the extent of direct physical contact with lipid double bonds, that is, DO15 had a 27-fold higher co-location with ω-9 lipid double bonds than methylene blue. By comparing the photoinduced effects of the dyes, they found that DO15 permeabilized membranes significantly faster than methylene blue, a result that was also valid for liposomes made of polyunsaturated lipids.

The researchers then identified and quantified all products generated by photosensitizers, such as hydroperoxides, alcohols, ketones and phospholipid aldehydes, in order to understand the mechanism by which DO15 was more efficient in permeabilizing the membranes. The most important finding was a significant increase in aldehyde production in the presence of DO15.

The importance of aldehyde formation for permeabilization was proven when the researchers irradiated liposome containing methylene blue for an extended time. In this condition, they observed that membrane permeabilization and aldehyde formation occurred to the same extent as in irradiated samples containing DO15 for a 40-fold shorter time period.

Although phospholipid aldehydes have been shown to disrupt chemical gradients - and hence increase membrane permeability - in membrane mimetic systems and in molecular dynamics simulations, they had not yet been detected in situ during photoinduced permeabilization of membranes.

"Our results are the first to definitively associate membrane permeabilization with the generation and accumulation of aldehyde in situ", said Baptista.

Another important result of the study was to verify that membrane permeabilization was invariably coupled to photobleaching of DO15, i.e., the compound was degraded.

In the development of new photosensitizers for medical applications, for example, the production of singlet oxygen has been considered the main parameter. However, the results of this work demonstrate that for a photosensitizer to totally compromise membrane function, it needs to be sacrificed through contact-dependent reactions. Thus, activation or suppression of regeneration of the photosensitizer could be explored as an effective tool to maximize or avoid the effects of photosensitized oxidations.

The article Photosensitized membrane permeabilization requires contact-dependent reactions between photosensitizer and lipids, by Isabel Bacellar, Maria Cecília Oliveira, Lucas Dantas, Elierge Costa, Helena Couto Junqueira, Waleska Kerllen Martins, Andrés M. Durantini, Gonzalo Cosa, Paolo Di Mascio, Mark Wainwright, Ronei Miotto, Rodrigo Maghdissian Cordeiro, Sayuri Miyamoto and Maurício S. Baptista, can be read at https://pubs.acs.org/doi/pdf/10.1021/jacs.8b05014