Molecular-Level Modifications Induced by Photo-Oxidation of Lipid

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On the molecular-level modifications induced by photooxidation of lipid monolayers interacting with erythrosin Pedro Henrique Benites Aoki, Luis F. C. Morato, Felippe José Pavinatto, Thatyane M. Nobre, Carlos Jose Leopoldo Constantino, and Osvaldo Novais Oliveira, Jr. Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00693 • Publication Date (Web): 27 Mar 2016 Downloaded from http://pubs.acs.org on March 27, 2016

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On the molecular-level modifications induced by photo-oxidation of lipid monolayers interacting with erythrosin Pedro H. B. Aoki1,2, Luis F. C. Morato3, Felippe J. Pavinatto1, Thatyane M. Nobre1, Carlos J. L. Constantino3 and Osvaldo N. Oliveira Jr1. 1

IFSC, São Carlos Institute of Physics, University of São Paulo (USP), São Carlos, SP, Brazil 13566-590 2

DCB, Faculdade de Ciências e Letras, UNESP Univ Estadual Paulista, Assis, SP, Brazil 19806-900

3

DF, Faculdade de Ciências e Tecnologia, UNESP Univ Estadual Paulista, Presidente Prudente, SP, Brazil 19060-900 Abstract Incorporation into cell membranes is key for the action of photosensitizers in photomedicine treatments, with hydroperoxidation as the prominent pathway of lipid oxidation. In this paper we use Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as cell membrane models to investigate adsorption of the photosensitizer erythrosin and its effect on photoinduced lipid oxidation. From surface pressure isotherms and polarization-modulated infrared reflectionabsorption spectroscopy (PM-IRRAS) data, erythrosin was found to adsorb mainly via electrostatic interaction with the choline in the head groups of both DOPC and DPPC. It caused larger monolayer expansion in DOPC, with possible penetration into the hydrophobic unsaturated chains, while penetration into the DPPC saturated chains was insignificant. Easier penetration is due to the less packed DOPC monolayer, in comparison to the more compact DPPC according to the monolayer compressibility data. Most importantly, light irradiation at 530 nm made the erythrosin-containing DOPC monolayer to become less unstable, with a relative surface area increase of ca. 19%, in agreement with previous findings for bioadhesive giant vesicles. The relative area increase is consistent with hydroperoxidation, supporting the

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erythrosin penetration into the lipid chains, which favors singlet oxygen generation close to double bonds, an important requirement for photodynamic efficiency. 1. Introduction Photodynamic therapy (PDT) has been efficient for treating different types of cancer1-3 and an alternative to chemical disinfection agents.4-7 It is based on the controlled administration of a photosensitizer (PS) that is further photo-activated in an environment surrounded by oxygen. Excited states of PS can transfer energy to molecular oxygen and form reactive oxygen species, such as singlet oxygen (1O2), hydroxyl radicals (OH•) and superoxide anion (O2−•). 1O2 is considered the most powerful cytotoxic agent in PDT, capable of causing necrosis or apoptosis in cancer cells.8-11 This has prompted a search for new PS with the ability to produce 1O2 at high quantum yield (ΦΔ),9,

12-13

though a high 1O2 ΦΔ is not always related to photodynamic

efficiency.14-17 PS molecules can be affected by interaction with the complex biological environment, and their spatial distribution defines where the oxidation species are generated. The relatively short 1O2 life time (3.5 µs in water) only allows it to diffuse over an average length of ld ~100 nm,18 which requires PS to be in the vicinity of the target (within ld) for 1O2 to act before decaying. Indeed, among PS with comparable 1O2 ΦΔ, those with affinity for biomolecules promote larger photodynamic damage. For instance, an enhanced efficiency of human cervical adenocarcinoma (HeLa) cell death was observed for a PS with high affinity for the mitochondrial membrane, despite its low 1O2 ΦΔ14. Therefore, unravelling the PS interaction mechanism to obtain improved selectivity in inducing cell death is key for understanding and controlling photooxidation in PDT. A controlled amount of oxidized lipids is required for different cell functions such as signaling, maturation, differentiation and cell apoptosis. On the other hand, uncontrolled lipid


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oxidation can lead to deleterious effects on cell functioning and might be associated with neurodegenerative diseases such as Parkinson and Alzheimer.19-20 The preferential sites of photodynamic action are the cell membranes,21 but the underlying mechanisms involved in membrane lipid oxidation are not fully understood. In order to investigate these mechanisms, suitable methods are required not only to mimic the cell membrane but also to obtain specific molecular information. Lipid monolayers assembled by the Langmuir technique are accepted as a model for half a membrane and have proven useful to determine mechanisms of action of many biologically-relevant materials.22-25 By way of illustration, Langmuir monolayers were useful for introduction of a novel concept of artificial enzymes (L-zyme) based on ester hydrolysis catalyzed by pK-tuned amino groups.26 In this work, Langmuir monolayers of the saturated 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) and unsaturated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were applied as membrane models to determine adsorption mechanisms of the photosensitizer erythrosin and the effects generated by lipid oxidation. Erythrosin belongs to the family of the xanthene derivatives, characterized by strong light absorption in the visible range and ability to initiate photochemical reactions.27-29 Surface pressure versus mean molecular area isotherms provided information on both erythrosin binding and membrane photo-oxidation. The molecularlevel interactions of erythrosin with lipid membranes were assessed by polarization-modulated infrared reflection−absorption spectroscopy (PM-IRRAS),30 a surface-specific spectroscopic technique able to identify changes in the orientation of chemical groups at the air-water interface.31 The use of PM-IRRAS was essential to unravel changes in the lipid polar head region and in the alkyl chains packing that follow membrane photo-oxidation.


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2. Experimental 2.1. Materials and solutions The zwitterionic phospholipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Avanti Polar Lipids Inc. The xanthene erythrosin B was acquired from Sigma-Aldrich Co. All chemicals were used without further purification. Ultrapure water (18.2 MΩ.cm) was provided by a Milli-Q system, model Simplicity, and used to prepare erythrosin solutions at 10-6, 10-5 and 10-4 mol/L. The molecular structures of DOPC, DPPC and erythrosin are given in Figure 1a.

2.2. Langmuir films Surface pressure versus area (π-A) isotherms were obtained in a KSV 2000 Langmuir through with the pressure being measured with the Wilhelmy plate method, where the plate was made of a filter paper. The phospholipid monolayers were prepared by dropping 1x10-3mol/L of chloroform stock solution (Merck, analytical grade) either onto ultrapure water (pH 5.8) or erythrosin (10-4, 10-5 and 10-6 mol/L) solutions. After spreading the phospholipid solution, the solvent was allowed to evaporate for ca. 15 min before the first compression. Langmuir monolayer compression was carried out at a constant barrier speed of 10 mm/min, keeping the water subphase temperature at 23ºC. No precaution to prevent oxidation was taken, but we confirmed that the surface pressure measured, at the same area per molecule, was within ± 2 mN/m. Results reported here represent average values of at least three different isotherms for each studied condition. It is known that exposure of unsaturated lipids to air can result in changes in the surface pressure in lipid monolayers.32 However, no significant change in surface pressure was observed during monolayer experiments since these effects were probably minimized by


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completing experiments relatively fast. The irradiation experiments were performed setting the monolayer surface pressure to 30 mN/m, and using a green LED (ca. 530 nm) source positioned 5 mm above the surface, as sketched in Figure 1b.

Figure 1: (a) molecular structures of DOPC, DPPC and erythrosin B. (b) Schematic representation of the Langmuir film irradiation setup. The LED was positioned 5 mm above the Langmuir monolayer, allowing the irradiation of the entire surface.

PM-IRRAS experiments were performed using a KSV PMI550 instrument (KSV, Finland). The light beam reached the monolayer with an incidence angle of 81o. Since the incident radiation was modulated between s and p polarizations at a high frequency and the spectra were obtained for both polarizations simultaneously, the effect of the water vapor was reduced. The difference between the s and p spectra provides information on the species present at the interface while the sum is the reference spectrum.33-34 Experiments were performed in phospholipid monolayers in the presence and absence of erythrosin with the surface pressure at 30 mNm−1. The reproducibility of band position was ensured to guarantee that the shifts observed are not related to spectra variability but to erythrosin interaction and further membrane photo-oxidation.


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3. Results and discussion 3.1. Erythrosin adsorption on DPPC and DOPC monolayers The π-A isotherms recorded for DPPC and DOPC Langmuir films on ultrapure water and erythrosin solutions at 10-4, 10-5 and 10-6 mol/L are shown in Figure 2a and 2b, respectively. In subsidiary experiments we observed that erythrosin molecules do not form Langmuir films on their own due to their high water solubility. The π-A isotherms are shifted toward larger molecular areas for increasing erythrosin concentration in the subphase, especially at and above 10-5 mol/L, with the shift remaining even at high surface pressures. In addition, the collapse pressure of DPPC monolayer increased by ca. 10 mN/m with the incorporation of erythrosin (Figure 2a). In spite of the structural similarity between the zwiterionic DPPC and DOPC phospholipids, the shifts in relative molecular area [(A-A0)/A0] as a function of erythrosin concentrations for DOPC π-A isotherms are approximately twice the values for DPPC, as shown the insets in Figure 2. This difference should be attributed to the unsaturation in the aliphatic chains of DOPC, which are less ordered than the saturated DPPC chains because DOPC has a lower melting-transition temperature (-17°C) than DPPC (41°C).32 The lower lipid packing of DOPC hydrocarbon tails35-36 should facilitate erythrosin incorporation, similarly to earlier reports for antimicrobial peptides.35


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Figure 2: π-A isotherms of (a) DPPC and (b) DOPC Langmuir films recorded at ultrapure water and erythrosin solutions at 10-4, 10-5 and 10-6 mol/L. The inset shows the relative mean molecular area displacement [(A-A0)/A0] as a function of erythrosin concentrations. A0 and A are the extrapolated areas at 30 mN/m of the isotherms recorded at ultrapure water and erythrosin solutions, respectively. The incorporation of erythrosin affected the monolayer elasticity, as shown in Figure 3 for the surface compressional modulus (Cs−1), or in-plane elasticity, calculated from the π-A isotherms in Figure 2. Cs−1 was taken as -A(∂π/∂A), where A and π are the molecular area and the surface pressure, respectively, at a given temperature.37 The maximum values of elasticity decreased from 268 to 247 mN/m for DPPC, and from 139 to 123 mN/m for DOPC, upon inserting erythrosin. Of particular relevance for biological implications, erythrosin reduced the monolayer elasticity at ca. 30 mN/m (insets of Figure 3), which is the surface pressure believed to correspond to the lateral pressure of cell membranes.38 This has important implications in pharmacology because the efficiency of some drugs may depend on the ability of modifying the membrane elasticity in order to facilitate mass transport across it, or even to compromise the integrity of the cell membrane.39


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Figure 3: in-plane elasticity (CS−1) calculated from (a) DPPC and (b) DOPC π-A isotherms using the expression: Cs−1 = -A(∂π/∂A). The insets show Cs−1 as a function of the surface pressure (π). The molecular-level interactions of erythrosin with the phospholipids in Langmuir monolayers can be assessed by PM-IRRAS, which is surface specific. The assignments of the main vibrational modes of DPPC and DOPC spectra in Figures 4 and 5 are shown in Table I along with the shifts caused by erythrosin and upon irradiation. The CH2 bands are shifted to higher wavenumbers in DOPC monolayers, owing to their more expanded character due to unsaturation in the alkyl chains, also noticed in the isotherms and elasticity graphs. According to Figure 4a, erythrosin affects the head group (left panel) significantly, but not the alkyl chains (right panel) of DPPC. The antisymmetric stretching of the choline group νas(CN+(CH3 )3) is shifted from 968 cm-1 to 954 cm-1 upon erythrosin interaction, suggesting that attractive electrostatic interactions occur with the negatively charged erythrosin40 being near the choline group (Figure 4b). The antisymmetric P=O stretching (νasPO2−) at 1234 cm−1 and the symmetric stretching (νsPO2−) band at 1080 cm−1 are shifted to 1219 (νasPO2−) and 1108 cm−1(νsPO2−), respectively. The band at 1051 cm-l assigned to (υ(C-O-PO2-)) stretching is shifted to 1076 cm-l for the DPPC monolayer containing erythrosin. These shifts in phosphate bands indicate that the interaction between erythrosin and DPPC molecules appears to have affected H-


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bonding with water molecules in the neighborhood. Phosphate groups have been proven amenable to H-bonding with water molecules,41 which may be affected by film disordering caused by erythrosin.42 The small changes induced by erythrosin in the C-H stretching of alkyl chains of DPPC are illustrated in the right panel in Figure 4a, which suggest no penetration (or very little) of erythrosin into the hydrophobic tail region. Nevertheless, the ratio between the peak intensity of the symmetric CH2 stretching (Is, ca. 2845 cm−1) and antisymmetric CH2 stretching (Ias, ca. 2918 cm−1) increased from 0.54 to 0.96 upon introducing erythrosin, thus indicating decreased order of the monolayer chains.43 This is consistent with the erythrosincontaining DPPC monolayer being more fluid than for neat DPPC, as inferred from the compressional modulus data (Figure 3). A model for the DPPC-erythrosin interaction based on the analysis of surface pressure and PM-IRRAS data is depicted in Figure 4b, where the positive and negative signs in phospholipid heads represent the choline and phosphate groups, respectively.


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Figure 4. (a) PM-IRRAS spectra of DPPC monolayers recorded at 30 mN/m with subphases of ultrapure water and erythrosin solution (10-5 mol/L). The left and right panel highlights the bands assigned to phosphate and alkyl chains, respectively. (b) Proposed model for DPPC-erythrosin interaction. The DPPC monolayer at air-water interface and under erythrosin influence is shown by the left and right panel, respectively. DPPC and DOPC molecules share the same phosphocholine head, which is the reason why one could expect rather similar interactions of erythrosin with the choline and phosphate groups. Indeed, the antisymmetric stretching of the choline group νas(CN+(CH3 )3) in DOPC monolayers is shifted from 977 cm-1 to 965 cm-1 upon erythrosin interaction, as shown in Figure 5a (left panel). The latter suggests that attractive electrostatic interactions bringing the negatively


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charged erythrosin nearby the choline group, similarly to what was found for DPPC. For neat DOPC monolayers the band assigned to the antisymmetric P=O stretching (νasPO2−) is at 1217 cm−1, while the band assigned to the symmetric stretching (νsPO2−) appears at 1088 cm−1. In the presence of erythrosin, the shape of the νsPO2− band at 1088 cm−1 was affected and the νasPO2− band at 1217 cm−1 was split into two bands at 1227 and 1263 cm−1. The band at 1062 cm-l for neat DOPC monolayer, assigned to the υ(C-O- PO2-) stretching, had the relative intensity affected compared with the band intensity at 1088 cm−1 (νsPO2−). Analogously to the effects on DPPC monolayers, the shifts in P=O stretching vibrations induced by erythrosin on DOPC molecules point to changes in H-bonding with water molecules. According to Zawisza et al.44, the νasPO2− band can be deconvoluted into two bands at 1227 and 1263 cm-1, as a result of weakly and strongly H-bonded PO2− in the monolayer. The increase in band intensity at 1263 cm-1 suggests that erythrosin induces strongly H-bonded PO2− group in the monolayer. The PMIRRAS signal of the carbonyl ester group stretching ν(C=O) of phospholipid molecules is centered at ca. 1730 cm-1.44 The exact position and the shape of this band depend on hydration of the polar region of the lipid and on polarity of the surrounding medium.45 For neat DOPC monolayers two bands are seen at 1714 and 1738 cm−1, corresponding to hydrated and nonhydrated carbonyl ester groups vibration, respectively. The bands within 1600 - 1700 cm−1 are due to the difference in reflectivity of the water interface covered and not covered with the monolayer. Upon erythrosin interaction the intensity of the hydrated ν(C=O) at 1714 cm−1 was significantly reduced while the nonhydrated ν(C=O) at 1738 cm−1 dominated the spectrum. Such modifications might be related to changes in the solvation of the carbonyl group.46 Therefore, erythrosin affects hydration nearby DOPC head groups, similarly to what has been reported for other photosensitizers46 and monovalent ions.47


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The bands assigned to the alkyl chains in the 2800−3000 cm−1 range at the right panel of Figure 5a appear at 2928 and 2860 cm-1 for antisymmetric νas(CH2) and symmetric νs(CH2) stretchings, respectively. Antisymmetric νas(CH3) stretching and CH stretching of HC=CH group are observed at 2952 cm-1 and 3002 cm-1. The incorporation of erythrosin affected these bands, as follows: (i) the Is/Ias ratio remains practically constant, but νas(CH2) and νs(CH2) stretchings were shifted to lower wavenumbers at 2918 and 2847 cm-1, respectively; (ii) the relative intensity of νas(CH3) at 2955 cm-1 increased; and (iii) υ(HC=CH) at 3002 cm-1 was shifted to 3015 cm-1. The shift in CH2 bands to lower wavenumbers points to an increased conformation order and packing of the alkyl chains.31 Overall, these spectral changes induced by erythrosin are more drastic for DOPC than for DPPC (factor (iii) is observed only for DOPC due to its unsaturation), especially considering the equipment spectral resolution (8 cm-1). Therefore, even though shifts in the CH2 bands could also arise from changes in chain packing and group orientation, one may presume that erythrosin penetrates into the hydrocarbon region of the DOPC monolayer, as depicted in Figure 5b. The latter corroborates the analysis of surface pressure isotherms and compressional modulus data.


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Figure 5. (a) PM-IRRAS spectra of DOPC monolayers recorded at 30 mN/m with subphases of ultrapure water and erythrosin solution (10-5 mol/L), with and without exposure to irradiation. The left and right panel highlights the bands assigned to the phosphate and alkyl chains, respectively. (b) Proposed model for DOPC-erythrosin interaction. The DOPC monolayer at airwater interface and under erythrosin influence is shown on the left and right panels, respectively.


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Table 1. Assignments of the main vibration bands of DPPC and DOPC Langmuir films along with the shifts induced by erythrosin interaction and photo-oxidation.44, 48 Discrepancies in these spectra in relation to published data for neat DOPC films should be attributed to differences in the experimental conditions used. DPPC (cm-1)

DOPC (cm-1)

Assignments

DPPC Neat

DPPC+ Erythrosin

DOPC Neat

DOPC + Erythrosin

DOPC + Erythrosin + Irradiation

2952 2918 2845 1735 1462 1234 1080 1051 968

2918 2850 1735 1470 1219 1108 1076 954

3002 2952 2928 2860 1738 1217 1088 1065 977

3015 2955 2918 2847 1738, 1714 1227, 1263 1088 1062 965

3010 2944 2911 2836 1738 1227 1082 1048 967

υ(HC=CH) υas(CH3) υas(CH2) υs(CH2) υ(C=O) δ (CH2) υas(PO2-) υs(PO2-) υ(C-O- PO2-) νas (CN+(CH3 )3)

3.2. Erythrosin photo-activation and membrane effects The effects of the membrane photo-oxidation are probed here by irradiating (ca. 530 nm) DPPC and DOPC monolayers in the presence of 10-5 mol/L erythrosin subphase. The surface area occupied by irradiated and non-irradiated monolayers was monitored over time at a constant surface pressure of 30 mN/m. The relative area evolution of DPPC monolayers (result not shown) was not affected under irradiation suggesting the absence of any major modification in membrane composition. On the other hand, significant changes were observed when the DOPC monolayer was irradiated. Figure 6a shows that the non-irradiated DOPC monolayers displayed a trend for decreasing area, probably owing to loss of material to the subphase. According to Liljeblad et al.,49 this loss might be related to lipid degradation by oxidation mediated by reactive species in the air. Upon irradiation, the slope of decrease in area is diminished, i.e. the monolayer became less unstable. This is clear in Figure 6b where the net effect, from erythrosin + irradiation, is plotted in the form of an increase in the area difference (∆).


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Under irradiation, excited states of erythrosin can transfer energy to molecular oxygen (O2) thus generating singlet oxygen (1O2), a very reactive oxygen species capable of diffusing over an average length of 100 nm before decaying. Pellosi et al.50 reported a high 1O2 quantum yield (ΦΔ ~ 0.62) for erythrosin. Since no significant modifications were observed for DPPC monolayers, interactions between 1O2 and DOPC unsaturations might be at the origin of the relative molecular area increase (∆). Reactions of 1O2 with unsaturated bonds of lipid chains generate hydroperoxide groups that further induce an increase in area per lipid due to modification of the carbon chain hydrophilic–hydrophobic balance.51-52 The hydroperoxide group is more hydrophilic than the lipid alkyl-chain, and may migrate toward the polar region of the monolayer,53-55 which would lead to a larger area occupied by the lipid molecule.52 The latter effect, in our case for DOPC, is illustrated by the inset in Figure 6b, where the relative molecular area for the DOPC irradiated film (relative to the non-irradiated film) increased with time, reaching a plateau around 20 min. A quantitative determination of area expansion in hydroperoxidized lipid membranes has been performed with a method based on the bio-adhesion of giant unilamellar vesicles (GUV), which yielded an area increase of ca. 18% for DOPC.56 Similar values were found by Weber et al.57 for DOPC GUV hydroperoxidation under micropipette aspiration. Even though the supramolecular arrangements differ between GUVs and Langmuir monolayers, it is remarkable that relative area increase (∆) under irradiation was ca. 19% in Figure 6b.


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Figure 6: (a) relative area (A/A0) evolution of irradiated and non-irradiated DOPC monolayers, in the presence of erythrosin, recorded at constant surface pressure of 30 mN/m. A0 is the extrapolated area at 30 mN/m of the DOPC isotherm. (b) Relative increase in mean molecular area of DOPC monolayer by taking the difference between irradiated and non-irradiated curves shown in (a). From the slope of the curve, the rate of relative area increase is 0.9 s-1. The inset depicts the DOPC hydroperoxidation generating an increase in the mean molecular area. Figure 5 shows that irradiation of DOPC monolayers in the presence of erythrosin causes significant changes in the PM-IRRAS for both head and tail groups, which does not occur for DPPC. In the head group, choline and phosphate groups were affected with νas(CN+(CH3 )3) being shifted from 965 cm-1 to 957 cm-1 upon irradiation. The υ(C-O-PO2-) stretching was shifted to 1048 cm-1 and the relative intensity of νs(PO2−) stretching at 1088 cm−1 increased. The relative intensity of νas(PO2−) stretching at 1227 cm−1 increased as well, and the band at 1263 cm-1 was no longer observed. Also significant was the increased intensity of bands at 1504 and 1529 cm-1, assigned to the xanthene ring stretching,58 which shows that erythrosin molecules coming from the subphase penetrate the irradiated DOPC monolayer. Moreover, the shifts for P=O stretching vibrations indicate that H-bonding between phosphate groups and water molecules in the neighborhood appears to be affected, also consistent with hydroperoxides being present at the monolayer-water interface. As for the tails, the νas(CH2) and νs(CH2) stretchings were shifted upon irradiation from 2918 and 2847 cm-1 to 2911 and 2836 cm-1, respectively. The relative


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intensity between νas(CH2) at 2918 cm-1 and νas(CH3) at 2955 cm-1 was affected and the υ(HC=CH) at 3015 cm-1 was shifted to 3010 cm-1. The changes in PM-IRRAS spectra induced by irradiation in erythrosin-containing DOPC monolayers point to modifications in packing, though the precise details of such modifications cannot be determined with the present results. They are nevertheless consistent with formation of hydroperoxide groups which occur when unsaturated chains are attacked by 1O2.31 The formation of hydrophilic hydroperoxide in the hydrophobic core of the monolayer may disrupt the van der Waals interactions responsible for stabilizing the lipid monolayer, altering membrane packing and affecting lipid-lipid interactions.59 Noteworthy is that the chain cleavage might take place due to propagation of the hydroperoxidation reaction, which could have generated truncated byproducts such as ketones, aldehydes or carboxylic acids.60-63 However, the presence of the υ(HC=CH) band after irradiation in Figure 5 indicates that the cleavage of the carbon chain near the initial position of the double bond does not occur, at least for the majority of DOPC molecules. We should note that the PM-IRRAS technique was not sufficiently sensitive to detect the hydroperoxide bands directly. This is why we stated that the results are consistent with (and not proof of) hydroperoxidation. In the search for further evidence of hydroperoxidation, we performed subsidiary experiments with giant unilamellar vesicles (GUVs) of DOPC containing 50 µM of erythrosin in the surrounding medium. They were irradiated at 547 nm and observed under phase contrast and fluorescence microscopies, simultaneously. Figure S1 in the Supplementary Material points to morphological changes being observed just after the beginning of irradiation. During the first ca. 10s, there was an increase in vesicle perimeter and projected area, and after ca. 2min of irradiation pore opening took place, which was followed by a drastic


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decrease in perimeter and vesicle collapse. These effects have been ascribed to hydroperoxide formation.51-52, 56-57, 62


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4. Conclusions Surface pressure isotherms and PM-IRRAS spectra indicated that the photosensitizer erythrosin could expand Langmuir monolayers of DOPC and DPPC, with stronger effect on DOPC. The lower density packing for the unsaturated DOPC monolayer may allow erythrosin insertion into the hydrophobic chains, thus favoring further lipid photo-oxidation via singletoxygen generation. The relative increase in surface area under irradiation and the modifications in the PM-IRRAS spectra are consistent with hydroperoxidation of the unsaturated chains, similarly to previous report on bioadhesive giant DOPC vesicles. In contrast, for erythrosincontaining DPPC monolayers irradiation did not cause any significant changes. Taken together, these results help explain the molecular-level mechanisms behind the photosensitizing action of erythrosin over membranes composed by unsaturated PCs, which includes mammalian plasma membranes. Acknowledgments This work was supported by FAPESP (2013/14262-7), CNPq and CAPES (Brazil). We thank prof. Dr. Carlos Marques and his team for the experiments involving GUVs.


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Molecular-Level Modifications Induced by Photo-Oxidation of Lipid

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