Methylene Blue Location in (Hydroperoxized) Cardiolipin Monolayer

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Methylene Blue Location in (Hydroperoxized) Cardiolipin Monolayer: Implication in Membrane Photodegradation R. M. de Souza,† P. Siani,† T. F. Schmidt,§,∥ R. Itri,‡ and L. G. Dias*,† †

Departamento de Química, FFCLRP, Universidade de São Paulo, Avenida Bandeirantes 3900, 14040-901, Ribeirão Preto, SP, Brazil Departamento de Física Aplicada, Instituto de Física, Universidade de São Paulo, Rua do Matão 187, 05508-900, São Paulo, SP, Brazil § Universidade Federal do ABC (UFABC), Avenida dos Estados 5001, 09210-580, Santo André, SP, Brazil ‡

S Supporting Information *

ABSTRACT: We present molecular dynamics simulations of cardiolipin (CL) and CL monohydroperoxized derivative (CLOOH) monolayers to investigate the initial steps of phospholipid oxidation induced by methylene blue (MB) photoexcitation under continuous illumination. We considered different MB atomic charge distributions to simulate the MB electronic distribution in the singlet ground and triplet excited states. Simulation results allied to experimental data revealed that initial CL photooxidation probably occurs via a type II mechanism, to produce lipid hydroperoxide by singlet oxygen attack to the alkyl chain unsaturations. The resulting hydroperoxide group prefers to reside near the aqueous interface, to increase the membrane surface area and to decrease lipid packing. Interestingly, MB orientation changes from nearly parallel to the water−monolayer interface in the ground state to normal to the interface in its triplet excited state. The latter orientation favors oxidative chain reaction continuity via a type I mechanism, during which the hydrogen atom must be transferred from the hydroperoxide group to triplet MB. Taken together, the present results can be extrapolated to improve our understanding of how oxidation progresses in lipidic biomembrane, which will lead to the formation of oxidized species with shortened chains and will cause severe photodamage to self-organized systems. μmol dm−3, DOPC GUVs evolve into large droplets until the vesicles are destroyed; above 100 μmol dm−3, the lipid bilayers follow an abrupt pathway that forms large membrane pores and disrupts the membrane. According to these investigations,18,20 such effects are linked to reaction of singlet oxygen molecules with DOPC unsaturated bonds, to produce oxidized lipid species and to reduce tail size.18 On the other hand, these investigations18,20 showed that low MB amounts increase the POPC and DOPC GUV membrane surface area, which is followed by contrast fading with irradiation time, an indication of increased membrane permeability.20 Riske et al.19 performed photodegradation experiments involving POPC GUVs and a photosensitizer based on a porphyrin molecule, whereas Weber et al.23 evaluated POPC and DOPC GUVs and a new photosensitizer based on a chlorin class of molecules. Both research teams showed that surface area increases rapidly due to a mild oxidation process that produces hydroperoxide from reaction between singlet oxygen and lipid unsaturated bonds. Interestingly, membrane integrity

1. INTRODUCTION Phenothiazines are heterocyclic molecules related to the thiazine class of compounds.1−3 Phenothiazines have long been used as insecticides and dyes, and they have been employed to treat infections caused by parasitic worms.1 In addition, phenothiazine derivatives such as promethazine and chlorpromazine constitute important antipsychotic drugs.1,4−6 Methylene blue (MB), a cationic phenothiazine derivative, has been widely used as a redox indicator. MB has also been employed to treat bacterial infection and tropical diseases, to disinfect blood, and to act as a photosensitizer during photodynamic therapy.7−17 Figure 1A illustrates the chemical structure of this compound. One of us (R.I.) has systematically explored MB (and other photosensitizers) as a photooxidizing agent (singlet oxygen quantum yield of 0.5) of biomembrane mimetic models18−23 by conducting studies on the photoresponse of membranes represented by 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-pamitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) giant unilamellar vesicles (GUVs) dispersed in aqueous solution containing different MB amounts.18,20 These investigations revealed that DOPC GUVs undergo degradation as a function of MB concentration:18 above 25 © 2017 American Chemical Society

Received: May 18, 2017 Revised: August 16, 2017 Published: August 18, 2017 8512

DOI: 10.1021/acs.jpcb.7b04824 J. Phys. Chem. B 2017, 121, 8512−8522

Article

The Journal of Physical Chemistry B

Figure 1. Chemical structures of (A) methylene blue (MB), (B) cardiolipin (CL), and (C) monohydroperoxized cardiolipin (CLOOH).

Figure 2. Schematic view of type I and type II photosensitization mechanisms.

peroxized lipid formation via a type II mechanism (i.e., triggered by singlet oxygen reaction with the lipid tail double bond) does not seem to modify membrane features

is preserved in both systems, but the membrane elastic modulus decreases by a factor of 4 for fully hydroperoxized membranes, while permeability remains unaltered.23 Therefore, hydro8513

DOI: 10.1021/acs.jpcb.7b04824 J. Phys. Chem. B 2017, 121, 8512−8522

Article

The Journal of Physical Chemistry B significantly, which could culminate in pore formation and membrane rupture. Figure 2 contains a schematic representation of type I and type II oxidation mechanisms. Bacelar et al.22 also observed membrane disruption when they compared how phenothiazinium derivatives (MB included) act during lecithin liposome photodamage. The authors suggested that a structure−property relationship may occur between the phenothiazium derivative hydrophobic− hydrophilic balance and the phenothiazium derivative photodisruption efficiency. They also suggested that type I and II mechanisms take place in the case of phenothiazium compounds, to push lipid oxidation beyond hydroperoxide species.21,22 Because MB has affinity for mitochondria,24 Schmidt et al.25 examined MB binding to Langmuir monolayers, mimicking the mitochondrion lipid composition. As expected, the authors found that MB adsorbs strongly onto 1,1′,2,2′-tetraoleoylcardiolipin (CL) monolayers through electrostatic interaction between the cationic MB and the anionic monolayer. It is worthy of note that GUVs composed of CL dispersed in low amount MB-containing aqueous solution also present phase contrast lost upon photoirradiation (Figure S3) similar to the effect previously observed for POPC and DOPC GUVs.20 Here, we revisit the MB−CL monolayer system from a computer simulation viewpoint. We also consider MB interaction with a CL monolayer monohydroperoxized version (Figure 1C). In particular, we explore how MB atomic charge distribution in the singlet fundamental state and in the triplet excited state is linked to MB localization in the monolayer and to the photosensitization mechanism (types I and II).

2. COMPUTATIONAL METHODOLOGY Molecular dynamics simulations were carried out in an all-atom representation for all species. The CL and the CL monohydroperoxized derivative headgroup parameters were obtained from Aguayo et al.26 The parameters were consistent with the CHARMM36 force field,27 from where ions and tail parameters were taken. The hydroperoxide group parameters were obtained from Garrec et al.,28 and the TIP3P29 water model was used. All of the MB bonds, angles, dihedrals, and Lennard-Jones parameters were also consistent with the CHARMM36 force field.30 The equilibrium geometry was fully relaxed in a vacuum at the B3LYP level31 with the def2-TZVPP basis set32 using C2v symmetry. Particular attention was paid to MB atomic charge distribution. A broad set of values has been reported in the literature.33−40 This work followed the procedure described in a recent work by Nunez et al.38 with slight modifications. Atomic charges were obtained in two different ways for the singlet ground state (S0): (i) single-point calculation in a vacuum and the CHELPG41 protocol at the Hartree−Fock (HF) level42 with the def2-TZVPP basis set;32 (ii) single-point calculation and the CHELPG protocol at the B3LYP/def2-TZVPP level in the COSMO water continuum model.43 These electronic structure calculations were performed with the software ORCAv.3.0.1.44 The second triplet excited state (T2) charges were obtained with the CHELPG protocol at the TDDFTB3LYP level with the 6-31+G* basis set45 after the geometry had been fully optimized with the software Gaussian 09.46 Figure 3 compares the charge distributions of the models (values for each atom can be found in the Supporting Information). Table 1 presents all of the dipole and quadrupole

Figure 3. Color representation of the atomic partial charge distribution. MB CHELPG atomic charges in the ground state (S0) as calculated at the (A) HF/def2-TZVPP level and (B) B3LYP(COSMO)/def2-TZVPP level and (C) in the second triplet excited state (T2) as calculated at the TDDFT-B3LYP/6-31+G* level.

Table 1. Total Dipole Moment and Traceless Quadrupole Moment Tensor Componentsa model

μz (D)

Qxx (DÅ)

Qyy (DÅ)

Qzz (DÅ)

MB() MB(T)

1.3 3.3 4.4

−52.2 −56.2 −50.8

80.3 91.0 83.2

−28.1 −34.8 −32.4

a In the Cartesian coordinate system, the x−y plane coincides with the MB molecular plane and the C2v(z) axis passes through this plane and is oriented from the nitrogen atom to the sulfur atom of the central ring.

components of the different charge models. Results referring to computation at the B3LYP and HF levels in S0 will be represented by the MB(>) and MB() fraction and a relatively smaller MB(