Elsevier

Dyes and Pigments

Volume 187, March 2021, 109131
Dyes and Pigments

Alkylation of a hydrophilic photosensitizer enhances the contact-dependent photo-induced oxidation of phospholipid membranes

https://doi.org/10.1016/j.dyepig.2020.109131Get rights and content

Highlights

  • O-decyl-Ptr acts mainly as contact-dependent photosensitizer and not by 1O2 formation.

  • O-decyl-Ptr binds to the membrane in a specific location, showed by MD simulations.

  • O-decyl-Ptr photo-induces oxidation in DOPC vesicles, producing LOOH, LO and LOH.

  • Cell viability is reduced by 50% when using O-decyl-Ptr as photosensitizer.

Abstract

Lipophilic photosensitizers able to photo-induce lipid oxidation in biomembranes are, in general, much more efficient than hydrophilic ones; acting through the formation of singlet oxygen (1O2), which oxidizes the fatty acid double bonds (type II mechanism). Here we investigate the binding and photosensitizing properties of 4-(decyloxy)pteridin-2-amine (O-decyl-Ptr) using unilamellar vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a phospholipid with monounsaturated fatty acids. By means of molecular dynamic (MD) simulations, we showed that O-decyl-Ptr binds to the membrane in a localization that favors the direct reaction of the triplet excited state of O-decyl-Ptr with DOPC double bonds. As a consequence, although 1O2 is formed, O-decyl-Ptr acts mainly as a contact-dependent photosensitizer, meaning through radical formation (type I mechanism). Mass spectrometry analysis of vesicles irradiated in the presence of O-decyl-Ptr, demonstrated the generation of alcohols (LOH), ketones (LO) and hydroperoxides (LOOH). In agreement with the mechanistic hypothesis proposed, LOH and LO (type I photooxidation products) are formed faster than LOOH (type II photooxidation product). Interestingly, no short-chain oxidized products were detected. Accordingly, membrane fluctuations and formation of filaments and buds are observed during in-situ photo-activation of O-decyl-Ptr in giant unilamellar vesicles due to changes in membrane spontaneous curvature. Finally, we evaluated the effect of the photochemical processes studied at a cellular level and demonstrated in experiments of viability of mammalian cells that O-decyl-Ptr has important photodynamic properties. Similar experiments performed using the hydrophilic photosenstizer pterin (Ptr) show that alkylation leads to a striking increase in the efficiency of photosensitized lipid oxidation.

Introduction

All unsaturated lipids in cell membranes, including phospholipids, glycolipids and cholesterol, are well-known targets of oxidative damage [1,2]. Lipid peroxidation can be produced by different mechanisms, for example, light-mediated processes such as photosensitized oxidation that naturally occurs and generate important membrane damage [3,4]. On the other hand, these processes can also have beneficial purposes such as the treatment of bacterial infections or different cancers, called photodynamic inactivation or therapy, respectively [[5], [6], [7], [8]].

Photosensitized oxidations can be triggered by two different mechanisms: i) a direct reaction of the triplet excited state of the photosensitizer with the target molecule, with the generation of radicals through electron transfer or hydrogen abstraction (type I mechanism), ii) via singlet oxygen (1O2) production by energy transfer from the triplet excited state of the photosensitizer to dissolved oxygen (type II mechanism) [9,10]. The first mechanism corresponds to contact-dependent pathways, and the latter to contact-independent pathways, where an intermediary species, 1O2, reacts with the target [11,12]. When the biological target is the lipid membrane, the photosensitizer can intercalate in the membrane (lipophilic compound) or remain in the aqueous phase (hydrophilic compound). In principle, one can guess that for lipophilic photosensitizers the contact-dependent pathways are predominant, whereas a competition between contact-dependent and –independent pathways takes place for hydrophilic photosensitizers. In this latter case, the predominant mechanism will depend on the photochemical properties of the photosensitizer.

The pterin moiety is present in the structure of the folic acid, being the main responsible for the photo-induced activity of this vitamin. Photodegradation of folic acid is one of the first and most devastating consequences of the skin over-exposure to the sun [13]. As such, pterins are endogenous chromophores involved in several depigmentation skin disorders, such as vitiligo [14,15]. Unfortunately, little is known about their photochemical properties in the skin. The comprehension of the relationship between the chemical structure of pterins and their molecular mechanisms of photodamage will allow the understanding of their harmful effects in human skin and consequently help in the design of better skin care agents. Also, pterin derivatives could work as photodynamic therapy agents in the photoinactivation of pathogenic microorganisms.

We have recently described the synthesis of new lipophilic photosensitizers derived from pterin (Ptr, Fig. 1), the parent member of the pterin family, which is a natural occurring compound and absorbs in the UVA region (320–400 nm) (Figure S1 of the Supplementary Material) [16]. These new lipophilic pterin derivatives have a decyl carbon chain attached which does not changes significantly the absorption spectrum (Figure S1), but favors their binding to the lipid membranes. Moreover, one of the conjugated pterins, 4-(decyloxy)pteridin-2-amine (O-decyl-Ptr, Fig. 1), was able to photo-oxidize polyunsaturated fatty acids (PUFAs) –containing lipid membranes upon UVA irradiation [17]. The efficiency on the photosensitized oxidation of lipids was notably higher for O-decyl-Ptr compared to Ptr, which freely passes across the membrane [18]. This difference was attributed to the fact that the binding of O-decyl-Ptr to the lipid bilayer strikingly increases the rate of contact-dependent processes. Oxidized lipids, such as hydroxy derivatives, hydroperoxides, and hydroxyhydroperoxides, were detected at the beginning of the photosensitized process. In the case of O-decyl Ptr, oxidized products quickly experience a cleavage of the fatty acid chains forming short-chain secondary products, which are responsible for the increase in the membrane permeability.

In this work we present a study of the contact-dependent photo-induced lipid peroxidation, using O-decyl-Ptr as a model of alkylated photosensitizers. As a substrate, we use 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, Fig. 1) to form unilamellar vesicles. DOPC is a phospholipid containing monounsaturated fatty acids (MUFAs), which are much less reactive than PUFAs and form a simpler set of oxidized products. Therefore, we chose DOPC as a substrate to: i) evaluate the capability of Ptr and decyl-pterins to photo-induce oxidation in MUFAs-containing membranes; ii) better analyze the mechanism and evolution of oxidized products. In particular, we have investigated the photosensitizer location into the lipid bilayer by means of molecular dynamic (MD) simulations, the products of the photosensitized oxidation and its consequences on mimetic membrane, features by means of mass spectrometry and optical microscopy of giant unilamellar vesicles (GUVs), respectively. Additionally, the effect of the photo-induced lipid peroxidation with O-decyl-Ptr at cellular level was assessed in mammalian cells.

Section snippets

Chemicals

Pterin (purity > 99%) was acquired from Schircks Laboratories (Switzerland) and O-decyl-Ptr was synthesized as reported earlier [16]. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, powder, ≥ 99%) was purchased from Avanti Polar Lipids Inc.

Computational methods

To evaluate the interaction with the biological membrane, the force fields for the molecule O-decyl-Ptr and Ptr were parameterized. Bonded and unbonded atom types and parameters were automatically determined using the CHARMM General Force Field (CGENFF) [19,20

Interaction studies with MD simulations

The affinity of O-decyl-Ptr to different lipid membranes was previously studied obtaining, in all cases, binding constant values of about 5 × 104 M−1 [16,17]. Decyl-pterins can be considered as amphiphilic compounds, and their chemical structure (Fig. 1) can be divided into a hydrophilic part (the pterin moiety) and a lipophilic part (the alkyl chain). The pterin moiety can be assumed as hydrophilic since Ptr is moderately soluble in water, very poorly soluble in organic solvents and does not

Conclusions

The contact-dependent photo-induced lipid peroxidation by 4-(decyloxy)pteridin-2-amine (O-decyl-Ptr), as a model of lipophilic photosensitizer, was investigated. Unilamellar vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a phospholipid containing monounsaturated fatty acids (MUFAs), were used as substrates. The results show that O-decyl-Ptr is able to photo-induce oxidation in MUFAs-containing membranes, generating not only hydroperoxides (LOOH), but also, and in higher amounts,

CRediT authorship contribution statement

Alejandro Vignoni: Investigation, Methodology, Software, Formal analysis. Carla Layana: Investigation, Methodology, Formal analysis. Helena C. Junqueira: Investigation. Andrés H. Thomas: Writing, Visualization, Resources. Rosangela Itri: Writing, Resources. Mauricio S. Baptista: Writing, Resources. Mariana Vignoni: Investigation, Methodology, Formal analysis, Writing, Project administration.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was partially supported by ANPCyT (Grants PICT 2016–1189 and 2017–0925), CONICET (PIP 2017–0274 and P-UE 2017 22920170100100CO), UNLP (Grant 11/X840), FAPESP (Process 2012/50680–5 and 2013/07937–8), Spanish MINECO/AEI and EU (Grant DPI2017-82896-C2-1-R) and Universitat Politècnica de València. M.V. thanks FAPESP for the Visiting Researcher Grant (2016/04296–0 and 2019/12957–4). The authors gratefully acknowledge the Scientific Computing Facility RIGEL from UPV, where MD simulation

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