1 Chain cleavage of bioinspired bacterial membranes photoinduced by eosin decyl ester Lucas G. Moreira1, Alexandre M. Almeida Jr.1, Sabrina A. Camacho1,2, Bianca M. Estevão2,3, Osvaldo N. Oliveira Jr.2, Pedro H. B. Aoki1 1 School of Sciences, Humanities and Languages, São Paulo State University (UNESP), Assis, SP, 19806-900, Brazil 2 IFSC, São Carlos Institute of Physics, University of São Paulo (USP), São Carlos, SP 13566-590, Brazil 3 Department of Chemistry, State University of Maringá (UEM), Maringá, PR, 87020- 900, Brazil 2 Abstract Photodynamic therapy (PDT) is promising for bacterial inactivation since cellular internalization of photosensitizers (PS) is not crucial for the treatment effectiveness. Photoinduced damage in the lipid envelope may already induce microbial inactivation, which requires PS capable of easily penetrating into the membrane. Herein, we report on the insertion of the PS eosin decyl ester (EosDec) into Langmuir films of 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac- glycerol) (DOPG) and cardiolipin (CLP) used as mimetic systems of bacterial membranes. Surface pressure isotherms and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) indicated that the hydrophobic nature of EosDec favored deeper penetration in all the phospholipid monolayers. The incorporation of EosDec led to monolayer expansion, especially in the anionic DOPG and CLP owing to repulsive electrostatic interactions, and induced disorder in the lipid chains. Light irradiation of DOPE, DOPG and CLP monolayers containing EosDec increased the rate of material loss to the subphase, which is attributed to cleavage of lipid chains triggered by contact-dependent reactions between excited states of EosDec and lipid unsaturations. The latter is key for membrane permeabilization and efficiency in microbial inactivation. Keywords: Langmuir monolayers, cell membranes, photosensitizers, photodynamic therapy, bacterial inactivation 3 Introduction Infectious diseases caused by drug-resistant microorganisms are major concerns to public health and global economics1. Bacterial resistance may originate from pre-existing innate mechanisms and acquired genetic materials, and intensified by the indiscriminate use2 and improper disposal of antibiotics3. Gram-negative bacteria, in particular, have an outer membrane with lipopolysaccharides (LPS), which makes them less susceptible to the action of antibiotics4–6 and demand alternative therapeutics7–9. An alternative is photodynamic therapy (PDT) for which cell internalization is not mandatory for bacterial inactivation10–13. PDT is based on a local or systemic administration of a photosensitizer (PS), further irradiated with appropriate wavelength to produce reactive oxygen species (ROS), which are cytotoxic to biological tissues, DNA, organelles, proteins, and lipid membranes14–16. Basically, the PS absorbs energy to reach its excited singlet state (1PS*) and undergoes intersystem crossing to the triplet state (3PS*), from which ROS can be formed via type I or type II reactions17–19. Created from the energy transfer to the molecular oxygen (3O2), singlet oxygen (1O2) is the main result of type II reaction, relevant for PDT. The short lifetime restricts 1O2 diffusion and confines the oxidation reactions to the PS vicinity. Therefore, PS location in the biological target is key to the photooxidation reactions and to the PDT success. Cell membranes are the main targets of 1O2 attack, which may lead to lipid hydroperoxidation as a result of ene reactions with alkenes containing allylic hydrogens20–22. Since phosphatidylcholines (PC) are major components of healthy eukaryotic cell membranes,23–27 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 increase21,22. We have hypothesized that the PS binding site can modulate the photochemical outcome from hydroperoxidation to the chain cleavage and 4 membrane permeabilization, triggered by contact-dependent reactions with excited states of PSs that penetrate deeper into the monolayer28,29. However, the limited penetration of water-soluble PS into the monolayers do not afford control over such modulation, which has prompted a search for PSs whose increased hydrophobicity allows effective insertion towards the lipid chains17,30. 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∆) for 1O2 17,31. The quantum yield is maintained owing to the halogen substitutes31 that increase the yield of intersystem crossing to 3PS* 32,33. For instance, eosin decyl ester (EosDec) has high Φ∆ 1O2 (0.38), excitation under visible light (λ=526 nm) and partition coefficient (1.84) of hydrophobic compounds17. Herein, we probe the molecular-level effects of the EosDec insertion into simplified model systems of bacterial membranes made with Langmuir monolayers of 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-snglycero-3-phospho-1'-rac-glycerol (DOPG) and cardiolipin (CLP). Photooxidation effects were investigated using surface pressure (π) vs mean molecular area (A) isotherms and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). As we shall demonstrate, the hydrophobic character of EosDec is key for deeper penetration into the monolayers and contact-dependent processes that yield chain cleavage and possible membrane permeabilization. 5 Experimental Section Materials The phospholipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) and cardiolipin (CLP, > 99%) were acquired from Avanti Polar Lipids. The photosensitizer eosin decyl ester (EosDec) was synthesized following previous-established protocols17. The molecular structures of DOPE, DOPG, CLP and the xanthene derivative EosDec are displayed in Figure 1a. The materials were used as received, with no further purification. Ultrapure water (resistivity = 18.2 MΩ·cm) from a Milli-Q equipment (model Direct-Q® 3UV) was employed as subphase in the Langmuir trough. Chloroform (CHCl3, 99,0 ~ 99,4%) from Sigma- Aldrich was used to dissolve the lipids and EosDec. Langmuir films fabrication 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 molecules22,28,34–39. 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 (23oC). 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. The solvent was allowed to evaporate for 15 min prior to the barriers compression at a constant rate of 5 mm/min. The surface pressure (π) versus mean molecular area (Å2) isotherms were recorded with the pressure measured using the Wilhelmy method with a platinum plate. No other procedure was adopted to avoid uncontrolled oxidation from the 6 environment33, but the reproducibility of the isotherms was ascertained by repeating the experiments and finding that the surface pressure varied within only ± 2 mN/m for a given area. Irradiation experiments The co-spread monolayers EosDec:DOPE (1:5), EosDec:DOPG (1:5) and EosDec:CLP (1:2) were irradiated at 30 mN/m with a LED source (ca. 530 nm, BRIWAX FFG/50 W) positioned 15 cm above the monolayer interface, as sketched in Figure 1b. Polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) was carried out in a KSV PMI550 (KSV, Finland) on neat monolayers of DOPE, DOPG, CLP and EosDec at 30 mN/m. The co-spread EosDec:DOPE (1:5), EosDec:DOPG (1:5) and EosDec:CLP (1:2) monolayers were also characterized, before and after irradiation. The experiments were performed from 1000 to 3100 cm-1 (incidence angle = 81° and resolution = 8 cm-1). The spectral reproducibility was checked by measuring the monolayers at least three times to ensure that the changes observed are not associated with spectral variability. Figure 1. (a) Molecular structures of DOPE, DOPG, CLP, and the xanthene derivative EosDec. (b) Illustration of the irradiation experiment. The LED is placed 15 cm above the co-spread film, allowing for irradiation of the entire surface. 7 Results and discussion EosDec incorporation into DOPE and DOPG monolayers The π-A isotherms for neat DOPE, DOPG, EosDec and the co-spread monolayers at 1:5 and 1:1 (EosDec:lipid) molecular ratios are displayed in Figure 2. The partition coefficient of 1.84 provides a hydrophobic character to EosDec17, which allows for Langmuir film formation with a high slope in the π-A isotherm and collapse pressure around 45 mN/m. The extrapolated area to zero pressure to the slope at 30 mN/m of 73 Å2 is small and suggests a highly packed monolayer owing to the π-π interactions between the xanthene rings40, which is similar to what was observed for the porphyrin derivatives ZnPP41, ZnPPIX-DME42 and Hemin43. The increase of EosDec molecular ratio up to 1:1 (EosDec:lipid) displaces the π-A isotherms to larger molecular areas due to the increasing amount of EosDec molecules incorporated by the phospholipid monolayers. Table 1 shows the change in relative molecular area [(𝐴 − 𝐴𝑜)/𝐴𝑜] as a function of EosDec molecular ratio. The change is larger for EosDec:DOPG than for EosDec:DOPE monolayers, independent of the EosDec amount. Both phospholipids have the same aliphatic chain, and therefore the polar head groups affect EosDec incorporation, probably because of repulsion with DOPG anionic polar groups which would not exist for the zwitterionic DOPE44–47. In the condensed phase, the slope of the isotherm for EosDec:DOPG (1:1) decreased, with a slight increase in the collapse pressure, which did not occur for EosDec:DOPE monolayers. The results for dianionic cardiolipin (CLP) monolayers are similar to those of DOPG, whose data and discussion are given as supplementary material (Figures S1 and S3). In addition, the mean molecular area vs the molar fraction of EosDec (XEosDec) 48,49 was analyzed for DOPE, DOPG and CLP co- spread monolayers (Figure S2 in the Supporting Information), confirming the monolayers expansion. 8 Figure 2. π-A isotherms of (a) DOPE, (b) DOPG and co-spread monolayers at 1:1 and 1:5 (EosDec:lipid) molecular ratio. The π-A isotherm of neat EosDec (black curve) is provided as control. Table 1. Relative shifts in area per phospholipid [( 𝐴 − 𝐴𝑜 𝐴𝑜 ) 𝑥 100)], where 𝐴𝑜 and 𝐴 are the extrapolated areas at 30 mN/m for the π-A isotherms of neat and co-spread monolayers, respectively. EosDec:phospholipid ratio Relative shift in the isotherm DOPE DOPG 1:1 70.2 % ± 0.9 % 85.7 % ± 2.8 % 1:5 8.5 % ± 2.1 % 11.0 % ± 1.5 % The molecular-level effects induced by EosDec incorporation into the lipid films were assessed here using polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). Table 2 brings the assignments of PM-IRRAS bands and displacements caused by EosDec incorporation and further irradiation. The spectra acquired for neat DOPE, EosDec and the co-spread EosDec:DOPE (1:5) monolayers are shown in Figure 3. (The PM-IRRAS data for CPL are discussed in the Supporting Information.) EosDec causes a minor effect over the DOPE head groups (Figure 3a) by increasing the band intensity at 1026 cm-1 and 1165 cm-1, assigned to υ(C–O–PO2 ─) and υas(C–O–C), respectively. The vibrational modes of the xanthene ring17,42 appear at 1520 cm-1 and 1450 cm-1, confirming the presence of EosDec in the monolayer. In phosphatidylethanolamine 9 membranes, hydrogen bonds between the phosphate and amine groups of adjacent molecules generate an inter-phospholipid network that decreases the availability of phosphates to establish new interactions50. Therefore, electrostatic interactions with the anionic EosDec are impaired, which is consistent with the slight modifications observed in the phosphate vibrational modes. As for the bands associated with the chains in Figure 3b, υs(CH2) at 2854 cm-1 is shifted to 2840 cm-1 with increased intensity while υas(CH3) at 2952 cm-1 had its intensity reduced upon EosDec incorporation. The ratio between the band intensity of the symmetric and antisymmetric CH2 stretching (Iυ𝑠(CH2)/Iυ𝑎𝑠(CH2)) increased from 0.50 to 1.08 indicating a decreased order of the chains51. This high impact on the tails may suggest penetration of EosDec molecules into the alkyl chains, as depicted in Figure 3c. Similar findings were observed for antimicrobial agents with amphiphilic character incorporated into saturated PE monolayers52,53. 10 Figure 3. PM-IRRAS spectra of the (a) polar heads and (b) alkyl tails acquired for neat DOPE, EosDec and co-spread EosDec:DOPE (1:5) films at 30 mN/m on aqueous subphase. (c) Proposed model for the interaction between EosDec – DOPE. The model is merely illustrative of the molecular-level interactions inferred from the monolayer characterization. The bands assigned to the phosphate groups are more disturbed in DOPG monolayers owing to the stronger electrostatic repulsion with the anionic EosDec. Figure 4a shows that the incorporation of EosDec shifts υ(C–O–PO2 ─) from 1057 cm-1 to 1023 cm-1, υas(PO2 -) from 1226 to 1246 cm-1 and increases υs(PO2 -) intensity at 1100 cm-1. It is likely that the interaction with EosDec has affected the hydrogen bonds with the surrounding water54,55, leading to dehydration of phosphate groups56. The C=C stretching mode at 1544 cm-1 is due to the xanthene ring and confirms EosDec incorporation into the DOPG monolayer. The C=C and C6H5 stretching modes at 1506 cm-1 and 1445 cm-1 in the neat EosDec spectrum shifted to 1483 cm-1 and 1433 cm-1 in the co-spread EosDec:DOPG 11 (1:5) film. The band position of the alky chains in Figure 4b remains unaltered except for the Iυ𝑠(CH2)/Iυ𝑎𝑠(CH2) ratio which increased from 0.30 to 1.23 owing to the disordering of the chains51. Considering these modifications with the larger areas in π-A isotherms (Table 1), one may conclude that DOPG monolayers are significantly more affected by EosDec incorporation. Electrostatic repulsion between the DOPG anionic portions have been increased by the insertion of anionic EosDec molecules, thus favoring the monolayer expansion and chain disordering, as indicated in Figure 4c. Figure 4. PM-IRRAS spectra of the (a) polar heads and (b) alkyl tails acquired for neat DOPG, EosDec and co-spread EosDec:DOPG (1:5) films at 30 mN/m on aqueous subphase. (c) Proposed model for the interaction between EosDec – DOPG. As in Figure 3(c), this schematic drawing is a mere illustration of the interactions. Table 2. Assignments of the main bands of neat DOPE, DOPG, EosDec and the co-spread EosDec:DOPE (1:5), EosDec:DOPG (1:5) Langmuir monolayers. 12 Assignments DOPE (cm-1) DOPG (cm-1) EosDec (cm-1) Water 1:5 Irrad Water 1:5 Irrad Water υ(HC=CH) 3017 3009 3009 3004 2997 3008 - υas(CH3) 2952 2952 2952 2956 2956 2975 2958 υas(CH2) 2924 2932 2924 2914 2917 2917 2921 υs(CH3) 2886 2892 2892 - - - - υs(CH2) 2854 2840 2862 2854 2846 2846 2850 υ(C=O) 1740 1740 1740 1738 1738 1745 1720 υ(C=C, Aromatic)2 - 1565 1565 - 1544 1542/ 1565 1558 υ(C=C, Aromatic)1 - 1520 1506 - 1483 1470 1506 υ(C6H5) - 1453 1453 - 1433 1434 1445 υ(C–O)2 - 1274 1274 - - - 1267 υas(PO2 -) 1227 1219 1219 1226 1246 1246 - υ(C–O)1 - - - - 1199 1206 1198 υas(C–O–C) 1165 1169 1169 1133 1133 1133 - υs(PO2 -) 1087 1087 1087 1100 1096 1096 - υ(C–O–PO2 -) 1026 1026 1026 1057 1023 1023 - *1:5 and 1:2 = EosDec:lipid; Irrad = Irradiated film EosDec photoactivation Photochemical reactions were monitored by measuring the surface area evolution of irradiated EosDec:DOPE (1:5), EosDec:DOPG (1:5) co-spread monolayers kept at 30 mN/m, as shown in Figure 5a and 5b. From subsidiary experiments we observed that irradiation has no effect on neat DOPE, DOPG and CLP monolayers, whose profiles do not statistically differ from the nonirradiated EosDec containing monolayers. A decrease in surface area is observed for all nonirradiated monolayers and is associated with the loss of material to the subphase generated by uncontrolled oxidation via reactive oxygen species (ROS) from the environment33,57. The anionic nature of DOPG increases 13 monolayer instability and the material loss rate, which is consistent with previous findings58. The slope of the irradiated monolayers is higher than for nonirradiated monolayers suggesting an enhanced material loss to the subphase, which was also noted for ozone oxidation59,60 and UV damage58. The higher stability of nonirradiated DOPE monolayer is the origin of the largest relative decrease in area, which at the end of 100 min of irradiation is reduced by 28 % ± 6% against 19% ± 5% for DOPG monolayers. As expected, the results for the negatively charged CLP in Figure S4 are similar to those of DOPG. The energy transfer from excited states of EosDec to the molecular oxygens (O2) generates singlet oxygen (1O2) species that can further react with lipid unsaturations. We have already shown that the tailless eosin Y adsorbs on phosphatidylcholine moieties by electrostatic interactions, with limited penetration into the chain region22. Upon irradiation, the 1O2 created in the vicinity of the hydrophilic groups were able to react with the chain unsaturation and form hydroperoxides. 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 reactions20–22,28. 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 hydroperoxides28. 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 1O2 be produced nearby the unsaturated region, and consequently favor phospholipid hydroperoxidation, but also the proximity of PS molecules would enable the generation 14 of aldehydes by contact-dependent processes, as shown in Figure 6. The latter induce cleavage of lipid tails, which may be the source of material loss in all monolayers. Indeed, the modifications in the PM-IRRAS spectra of irradiated monolayers in Figure 5 are consistent with such oxidative reactions. For instance, the panel on the right of Figure 5a shows that the υ(C–O–PO2 ─) band intensity at 1026 cm-1 is reduced on the irradiated EosDec:DOPE (1:5). Moreover, the bands due to carbonyl ester groups at 1169 cm-1 and 1740 cm-1 had their intensity increased, indicating modifications in the hydration of the lipid polar region61. The vibrational modes of the xanthene rings were also affected with a decreased intensity of the phenyl group at 1429 and 1453 cm-1. In addition, the υ(C=C, Aromatic)1 band at 1520 cm-1 shifted to 1506 cm-1 and the intensity of υ(C=C, Aromatic)2 at 1565 cm-1 increased17,42. As for the tails, the υs(CH2) band at 2840 cm-1 shifted to 2862 cm-1 with decreased intensity while υas (CH3) at 2952 cm-1 had the intensity significantly increased. The Iυ𝑠(CH2)/Iυ𝑎𝑠(CH2) ratio was barely modified suggesting that the chain disorder induced by EosDec insertion was maintained upon irradiation. Figure 5. Relative area (A/A0) progress of the nonirradiated (black curves) and irradiated (red curves) (a) EosDec:DOPE (1:5) and (b) EosDec:DOPG (1:5) monolayers. A0 is the 15 extrapolated area of the phospholipid isotherms at 30 mN/m down to zero pressure. The PM-IRRAs spectra recorded for nonirradiated and irradiated co-spread monolayers are provided in the panel on the right. Stronger modifications were observed in the phosphate region of irradiated EosDec:DOPG (1:5) monolayers, shown in Figure 5b. The intensity of υ(C–O–PO2 ─) band at 1023 cm-1 decreased while an increase was observed for υs(PO2 -) and υas(PO2 -) at 1096 and 1246 cm-1. In addition, the intensity of the carbonyl ester group band at 1738 cm-1 decreased. The xanthene vibrational modes were affected in a similar way to that for irradiated EosDec:DOPE (1:5) monolayer. The υ(C=C, aromatic)1 band shifted from 1483 to 1470 cm-1, the υ(C=C, aromatic)2 band at 1544 cm-1 split into two bands at 1542 and 1565 cm-1, and the C6H5 stretching band at 1433 cm-1 had its intensity reduced17,42. Such modifications are consistent with the report on lipid hydroperoxidation22, which can impact the organization of EosDec within the monolayer. The chains were also affected with an increase in the υas(CH2) band at 2917 cm-1, resulting in a slight decrease in the chains disorder with a reduced Iυ𝑠(CH2)/Iυ𝑎𝑠(CH2) ratio51. Besides, the υas(CH3) band shifted from 2956 to 2975 cm-1 and the υ(HC=CH) band shifted from 2997 to 3008 cm-1. The presence of υ(HC=CH) band in both EosDec:DOPE and EosDec:DOPG irradiated monolayers suggests the solubilization of the cleaved lipids in the subphase, leaving the uncleaved lipids at the interface to be probed. 16 Figure 6. Scheme of the alkyl chains cleavage under contact reactions between excited states of EosDec and unsaturations. 17 Conclusions Langmuir films of DOPE, DOPG and CLP phospholipids were used here as simplified models of bacterial cell membranes to unravel the molecular-level interactions with the photosensitizer eosin decyl ester (EosDec) and further photoinduced effects. EosDec incorporation affected the polar heads region, especially for the anionic DOPG and CLP owing to electrostatic repulsion that led to larger monolayer expansion. Furthermore, the hydrophobic nature of EosDec allowed deeper penetration into the monolayers causing an increase in chain disorder. The photoactivation of the incorporated EosDec decreased the stability of DOPE, DOPG and CLP monolayers, and induced an increased loss of material to the subphase, bringing chemical and physical disturbances to the monolayer. The penetration of EosDec into the hydrophobic core of the monolayer favored contact-dependent reactions between the excited triplet state of EosDec and lipid unsaturations or hydroperoxides previously formed, resulting in the cleavage of the chains. These findings contribute to the understanding on how the molecular-level interactions with photosensitizers impact the photosensitization outcome in model systems of bacterial membranes, which are relevant for photodynamic therapy in microbial inactivation. Acknowledgments This work was supported by São Paulo Research Foundation (FAPESP 2013/14262-7, 2018/16713-0, 2018/14692-5 and 2018/22214-6), INEO and National Council for Scientific and Technological Development (CNPq Universal 403713/2016-1). We thank Diogo S. Pellosi and Dr. Wilker Caetano for their assistance with EosDec. Lucas Gontijo Moreira is thankful for his scholarship provided by FAPESP (2018/13021-0). 18 Conflicts of interest There are no conflicts to declare. 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