Role of Toluidine Blue-O Binding Mechanism for Photooxidation in Bioinspired Bacterial Membranes

Abstract

The alarming increase in bacterial resistance to antibiotics has demanded new strategies for microbial inactivation, which include photodynamic therapy whose activity relies on the photoreaction damage to the microorganism membrane. Herein, the binding mechanisms of the photosensitizer toluidine blue-O (TBO) on simplified models of bacterial membrane with Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG) were correlated to the effects of the photoinduced lipid oxidation. Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were also used as a reference of mammalian membranes. The surface pressure isotherms combined with polarization-modulated infrared reflection absorption spectroscopy revealed that TBO expands DOPC, DOPE, and DOPG monolayers owing to electrostatic interactions with the negatively charged groups in the phospholipids, with a stronger adsorption on DOPG, which has a net surface charge. Light irradiation made the TBO-containing DOPC and DOPE monolayers less unstable as a result of the singlet oxygen ( 1 O 2 ) reaction with the chain unsaturation and hydroperoxide formation. In contrast, the decreased stability of the irradiated TBO-containing DOPG monolayer suggests the cleavage of carbon chains. The anionic nature of DOPG allowed a deeper penetration of TBO into the chain region, favoring contact-dependent reactions between the excited triplet state of TBO and lipid unsaturations or/and hydroperoxide groups, which is the key for the cleavage reactions and further membrane permeabilization.

Chain Cleavage of Bioinspired Bacterial Membranes Photoinduced by Eosin Decyl Ester

“…The proximity with the chains allowed the permeabilization of the membranes by contact-dependent reactions between excited states of TBO and unsaturations or previously formed hydroperoxides. 28 The hydrophobic character of EosDec allows insertion into the aliphatic chains of the phospholipids, as inferred from the PM-IRRAS data. Therefore, not only would 1 O 2 be produced nearby the unsaturated region, and consequently favor phospholipid hydroperoxidation, but also the proximity of PS molecules would enable the generation of aldehydes by contact-dependent processes, as shown in Figure 6.…”

“…Their hydrophilic character led these groups toward the water interface, resulting in the overall surface area increase of the monolayer. There is also evidence that molecular-level interactions of photosensitizers with lipid monolayers modulate the photochemical reactions. − , For instance, deeper penetration of the cationic toluidine blue-O (TBO) into the chain region of phosphatidylglycerol monolayers was observed owing to stronger attractive electrostatic interactions. The proximity with the chains allowed the permeabilization of the membranes by contact-dependent reactions between excited states of TBO and unsaturations or previously formed hydroperoxides …”

“…Langmuir monolayers of phospholipids may be used as simplified models of only half of a cell membrane. They are nevertheless useful since the lateral packing and membrane composition can be controlled, and molecular-level interaction can be inferred with in situ measurements, including with PM-IRRAS which permitted determination of the action mechanism of biologically relevant molecules. ,,− The Langmuir films were fabricated in a Langmuir trough (KSV-NIMA/KN 2002) by spreading each phospholipid solution (10 –3 mol/L) on an aqueous subphase at room temperature (23 °C). The same methodology was applied for neat EosDec, whereas for the EosDec/lipid films we first spread the phospholipid solution and then the EosDec solution immediately afterward.…”

“…Cell membranes are the main targets of 1 O 2 attack, which may lead to lipid hydroperoxidation as a result of ene reactions with alkenes containing allylic hydrogens. − Since phosphatidylcholines (PC) are major components of healthy eukaryotic cell membranes, − we have used them in Langmuir monolayers as simplified models and showed that hydrophilic hydroperoxides created in the hydrophobic core of phospholipids disrupt the hydrophilic–hydrophobic balance of the monolayer, leading to an overall surface area increase. , We have hypothesized that the PS binding site can modulate the photochemical outcome from hydroperoxidation to the chain cleavage and membrane permeabilization, triggered by contact-dependent reactions with excited states of PSs that penetrate deeper into the monolayer. , However, the limited penetration of water-soluble PS into the monolayers does not afford control over such modulation, which has prompted a search for PSs whose increased hydrophobicity allows effective insertion toward the lipid chains. , …”

“…21,22 We have hypothesized that the PS binding site can modulate the photochemical outcome from hydroperoxidation to the chain cleavage and membrane permeabilization, triggered by contact-dependent reactions with excited states of PSs that penetrate deeper into the monolayer. 28,29 However, the limited penetration of water-soluble PS into the monolayers does not afford control over such modulation, which has prompted a search for PSs whose increased hydrophobicity allows effective insertion toward the lipid chains. 17,30 This challenge has been addressed with the synthesis of xanthene ester derivatives containing alkyl groups, which keep their light absorption, low toxicity, and high quantum yield (Φ Δ ) for 1 O 2 .…”