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The interaction of eugenol with cell membrane models at the air–water interface is modulated by the lipid monolayer composition
Biophysical Chemistry 207 (2015) 7–12
Contents lists available at ScienceDirect
Biophysical Chemistry journal homepage: http://www.elsevier.com/locate/biophyschem
The interaction of eugenol with cell membrane models at the air–water interface is modulated by the lipid monolayer composition Giulia E.G. Gonçalves, Fernanda S. de Souza, João Henrique G. Lago, Luciano Caseli ⁎ Institute of Environmental, Chemical, and Pharmaceutical Sciences, Federal University of São Paulo (UNIFESP), Diadema, São Paulo, Brazil
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Langmuir monolayers of lipids are formed at the air–water interface. • Eugenol is incorporated to the monolayer. • Interactions at the molecular level are identified with tensiometry and PM–IRRAS. • Intermolecular interactions are modulated by the monolayer lipid composition.
a r t i c l e
i n f o
Article history: Received 3 July 2015 Received in revised form 21 July 2015 Accepted 26 July 2015 Available online 29 July 2015 Keywords: Eugenol Langmuir monolayer Air–water interface Biomembranes Drug
a b s t r a c t Eugenol, a natural phenylpropanoid derivative with possible action in biological surfaces as microbicide, anesthetic and antioxidant, was incorporated in lipid monolayers of selected lipids at the air–water interface, representing cell membrane models. Interaction of eugenol with the lipids dipalmitoylphosphatidylcholine (DPPC), dioctadecyldimethylammonium bromide (DODAB), and dipalmitoylphosphatidylserine (DPPS) could be inferred by means of surface pressure-area isotherms and Polarization–Modulation Reflection–Absorption Spectroscopy. The interaction showed different effects on the different lipids. A higher monolayer expansion was observed for DPPS and DODAB, while more significant effects on the polar groups of the lipids were observed for DPPS and DPPC. These results pointed to the fact that the interaction of eugenol with lipid monolayers at the air–water interface is modulated by the lipid composition, which may be important to comprehend at the molecular level the interaction of this drug with biological surfaces. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Eugenol is an allylphenol, belonging to the class of natural products known as phenylpropanoids. It is extracted from essential oils such as, ⁎ Corresponding author at: Rua São Nicolau, 210, Unifesp, Diadema São Paulo, 09913-030, Brazil. E-mail address: [email protected] (L. Caseli).
http://dx.doi.org/10.1016/j.bpc.2015.07.007 0301-4622/© 2015 Elsevier B.V. All rights reserved.
nutmeg, clove oil, cinnamon, basil and bay leaf [1–3]. This compound has been reported to be used in flavorings, perfumeries, and in medicine as local antiseptics and anesthetics. Also it is considered as a bactericide, and an antiviral compound [4, 5], and can be employed as restorative applications in dentistry, in antioxidants for plastics and rubbers, as an anesthetic, and in some mousetraps. Also it has been reported that eugenol kills certain human colon
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cancer cell lines in vitro and in vivo [6–8]. Furthermore, it is reported that it is hepatotoxic, causing damage to living beings [9]. In this sense, it is of interest to study the interactions of this compound in biointerfaces, such as cell membranes. The understanding of how eugenol interacts with these surfaces may help comprehend its molecular mechanism of action in cell membranes. It is reported that eugenol inserted in liposomes inhibits the enzymatic action of xanthine oxidase–xanthine-iron [10]. Also, it has been shown that eugenol prevents membrane damaging events for which its presence resulted in the inhibition for the formation of malondialdehyde in irradiated liposomes. Fujisawa et al. [11] reported that the cytotoxic activity of eugenol against cells was enhanced by visible-light irradiation due to its high redox potential. A proper system to be used as a model for cell membranes is the Langmuir monolayers, which is composed by monomolecular films of amphiphilic compounds at the liquid–gas interface. When organic solutions of membrane lipids are spread on the air–water interface, a model for half a membrane is formed [12], and interactions with proteins [13], polysaccharides [14], peptides [15] and drugs [16] can be investigated by means of tensiometry, vibrational spectroscopy and other surface specific techniques. Particularly, the interaction of several drugs with lipids at the air–water interface have been studied in Langmuir monolayers of lipids [16–18], and to the best of our knowledge no report on the interaction of eugenol with cell membrane models represented by a Langmuir monolayer has been reported. However, some studies have been shown the insertion of eugenol in bilayers [10–19], which makes promising the study with Langmuir monolayers as a complementary model. Also, these monomolecular films are useful to understand molecular interactions for other kind of lipid-drug systems, such as in liposomes in drug delivery systems. In this present work, we studied the interaction of eugenol with Langmuir monolayers composed of selected lipids. In order to better understand the role of the chemical nature of the lipid in these interactions, three different lipids were employed, a zwitterionic one, dipalmitoylphosphatidylcholine (DPPC), a positively charged one, dioctadecyldimethylammonium bromide (DODAB), and a negatively charged lipid, dipalmitoylphosphatidylserine (DPPS). 2. Materials and methods 2.1. General DPPC, DODAB and DPPS were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in chloroform (Synth, Diadema, Brazil) to a concentration of 0.5 mg/mL. The monolayer subphase approximated physiological conditions and consisted of a 50 mM phosphate buffer and 150 mM NaCl at a pH of 7.4. The water employed was purified using a MilliQ-Plus System (resistivity 18.2 MΩ cm, pH 5.5). LREIMS was measured in MS-QP-5050A mass spectrometer, operating at electron impact (70 eV). 1H and 13C NMR spectra were recorded, respectively, at 300 and 75 MHz in a Bruker Advance II 300 spectrometer using CDCl3 (TediaBrazil) as solvent and TMS (tetramethylsilane) as internal standard. Chemical shifts (δ) are reported in ppm and coupling content (J) in Hz. 2.2. Isolation of eugenol Aiming the isolation of eugenol, twigs of Nectranda leucantha were collected in the municipality of Cubatão, São Paulo State, Brazil in November 2013. The dried and powdered plant material (300 g) was exhaustively extracted with n-hexane, and its concentration was reduced under pressure, obtaining about 9 g of n-hexane extract. Part of this crude material (7 g) was subjected to column chromatography over silica gel using increasing amounts of EtOAc in n-hexane to afford six fractions (A to F). Fraction D (76 mg) was purified by Sephadex LH-20 using MeOH as eluent to afford 13 mg of pure eugenol.
Eugenol was characterized by NMR (300 and 75 MHz, CDCl3) and follows: δH 6.67 (d, J = 2.0 Hz, H-2), 6.84 (d, J = 8.1 Hz, H-5), 6.70 (dd, J = 8.1 and 2.0 Hz, H-6), 3.32 (d, J = 6.6 Hz, H-7), 5.93 (m, H-8), 5.06 (m, H-9), 3.87 (s, 3-OCH3). δC 131.9 (C-1), 111.1 (C-2), 146.5 (C3), 143.9 (C-4), 121.2 (C-5), 114.3 (C-6), 39.9 (C-7), 137.8 (C-8), 115.5 (C-9), 55.9 (3-OCH3). LREIMS m/z (rel. int.) 164 (M+, 100), 149 (47), 137 (31), 131 (40), 121 (20), 103 (25), 91 (20), 77 (21). 2.3. Preparation of monolayers The Langmuir monolayers were obtained by spreading a chloroform solution of DPPC, DPPS or DODAB on the surface of an aqueous buffer solution. For preliminary tests, eugenol solutions, dissolved in chloroform to a concentration of 0.5 mg/mL, were also spread alone at the air–water interface in order to test the surface activity of this compound. For mixed eugenol-lipid monolayers, first the lipid was spread on the air– water interface. Then, 20 min was allowed for chloroform evaporation, and aliquots of eugenol to render a 2% in mol-lipid were carefully injected in the aqueous subphase. Other proportions were essayed and this value represents a limit for the effect of this drug in terms of expansion of the monolayer. For higher amounts the isotherms do not present reproducibility probably because effects related to the formation of aggregates. Also, considering that drugs interacting with membranes are inserted in relative small amounts, this proportion must approximate conditions in vivo. Surface pressure-area (π-A) isotherms were obtained in a mini-KSV Langmuir trough equipped with a surface pressure sensor (Wilhelmy method), with an interface compression rate of 5 Å2 molecule−1 min−1. After allowing 20 min for evaporation of chloroform, the monolayer was compressed until the collapse is reached. Each isotherm shown in this paper was repeated at least three times for checking the reproducibility, and no variations higher than 0.1 mN/m was allowed. Then for each graph, a representative isotherm is shown. For PM–IRRAS studies, the monolayer was compressed until the desired surface pressure (30 mN/m). The surface pressure was maintained at the desired surface pressure by moving the barriers, and the stabilization of the monolayer was monitored until no additional movement of the barriers was needed. The PM–IRRAS measurements were then taken using a KSV PMI 550 instrument (KSV Instruments, Ltd., Helsinki, Finland) at a fixed incidence angle of 80°. Each spectrum shown in this paper was repeated at least three times for checking the reproducibility and a representative spectrum is shown. All experiments were carried out at a controlled room temperature (25 °C). 3. Results and discussion After isolation of eugenol from twigs of N. leucantha, its structure was defined by analysis with 1H, 13C NMR and LREIMS data and compared with data previously described in the literature [11,20]. Eugenol spread alone on the air–water interface in the concentration used in this work does not present any surface activity, i.e. its spreading on the air–water interface does not decrease the surface tension of water. When the interface is compressed, thus increasing the surface molecular density, the surface pressure does not raise more than 1 mN/m, which confirms that this compound does not form Langmuir monolayers. As they are insoluble in water and cannot leave the interface, this fact can be attributed to the low spreading coefficient of eugenol, which must cause its aggregation at the air–water interface. When incorporated in lipid monolayers, the surface activity of eugenol is detected, as inferred by means of analysis of the surface pressurearea isotherms. Fig. 1A shows the action of eugenol in Langmuir monolayers of DPPC. This lipid presents a typical π-A isotherm [21], with a plateau at approximately 14 mN/m, representing the transition between the states liquid-expanded (LE) and liquid-condensed (LC). The monolayer collapses at 55–60 mN/m in molecular areas of 45–50 Å2.
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indicates that the monolayer is less elastic to the compression, which may be associated to the ability of the monolayer to re-arrange molecularly upon the effect of lateral compression. It is important to mention that other proportions were essayed and the proportion in mol of 2% of eugenol in relation to the lipids represented a limit for the effect of this drug in terms of expansion of the monolayer. For higher amounts, the isotherms do not present reproducibility, probably owing to supramolecular effects such as the formation of aggregates. Lower amounts present similar effects until 0.5%, and below this value no significant effect was observed in the isotherms. Also, an evaluation of the possible loss of area monolayer was tested upon repetitive compression–expansion cycles. This was carried out until 5 cycles for all monolayers, pure or mixed, and no relevant hysteresis was observed. Fig. 2 shows the PM–IRRAS spectra for the monolayers at 30 mN/m. This value was chosen because it is related to the value of lateral pressure encountered in natural membranes [21]. Panel A shows the bands assigned for CH2 stretches. The ratio between the area below the curves for bands attributed to the antisymmetric (2915 cm−1) and symmetric (2848 cm−1) stretches is related to an order parameter [22]. For pure DPPC this value is 1.9; and for eugenol-DPPC it is 1.4. This decrease of order is closely related to the lower compressibility, indicating the effect of the drug. Also the band centered at 2878 cm−1, attributed to CH3 groups, is more evident for the mixed monolayer, whereas for the pure DPPC monolayer, it seems that this band appears as a shoulder.
Fig. 1. Surface pressure-area (A) and compressibility modulus-area (B) isotherms for DPPC and DPPC-eugenol (2% in mol) monolayers.
When mixed with eugenol, apparently no change is observed in the gaseous and liquid-expanded region (from 145 to 75 Å2). Further compression leads to a shift of the isotherm to higher areas in relation to the isotherm for pure DPPC. This fact is observed until the surface pressure of 32 mN/m. For higher surface pressures, the isotherm for the mixed film is shifted to lower molecular areas and collapse is observed at 50 mN/m. The shift to higher molecular area indicates the expansion of the lipid monolayer and can be associated to the incorporation of the drug inside the alkyl chains of the phospholipids. This fact may also change the transition of phases for DPPC since a transition typical for a pure substance (a plateau in the isotherm) is not well defined anymore. The shift of the isotherm to lower molecular areas indicates the condensation of the monolayer and may indicate either expulsion of the drug from the monolayer or molecular accommodation of the drug during the compression. As the surface pressure of collapse and the monolayer compressibility changed at such high surface pressures, it is likely that the second hypothesis is the most probable. It is important to emphasize that for these specific curves, the isotherms were repeated 5 times and no significant deviation (i.e. more than 0.1 mN/m) was observed, assuring the reproducibility of the experiments. Also, it is clear that the binary mixtures formed are not ideal, and then some changes in the isotherms at high surface pressures may be a consequence of the partial squeezing of eugenol from the monolayer or the self-segregation into enriched eugenol microdomains. Fig. 1B shows the compressibility modulus is lower for the mixed monolayer in relation to the pure lipid monolayer. This not only confirms the incorporation of eugenol at such high values of surface pressure, but also
Fig. 2. PM–IRRAS for DPPC and DPPC-eugenol (2% in mol) monolayers at 30 mN/m.
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Panel B for Fig. 2 shows the region for hydrophilic groups of DPPC. The band centered at 1723 cm−1 is attributed to carbonyl stretches, and is shifted to 1736 cm−1 upon incorporation of eugenol. The band centered at 1225 cm− 1 is attributed to phosphate stretches and is shifted to 1261 cm−1 owing to eugenol incorporation. The band centered in 1168 cm−1 may be attributed to ring vibrations of the drug. These results show that the drug may affect also the hydrophilic groups of DPPC. Fig. 3 shows the surface pressure-area isotherms for DPPS. With the presence of eugenol the isotherm is shifted to higher areas. This effect is more prominent than that observed for DPPC, and indicated noticeably the expansion of the monolayer, probably caused by the penetration of eugenol in the monolayer and to repulsions due to its presence. Eugenol is not charged in this pH, but DPPS is, so these repulsions must have origin from dipole–charge interactions. Due to its expansion, even the initial surface pressure begins in values above zero (about 10 mN/m). Also the rheological properties are affected since Fig. 3B shows significant changes in the compressibility of the monolayer. The LE–LC transition occurs for pure DPPS between the areas of 90 and 60 Å2 at 15–16 mN/m. With the presence of eugenol, this range occurs between 120 and 90 Å2 at 16–17 mN/m. These higher values of area are an effect of the monolayer expansion. For the mixed monolayer, another transition is observed between 80 and 70 Å2 at the surface pressure of 25 mN/m, and may be attributed to molecular accommodations of the drug embedded in the lipid monolayer during the compression. The maximum value of compressibility modulus (480 mN/m) is lower than for the pure lipid
monolayer (520 mN/m), and the collapse pressure decreases from 70 to 66 mN/m. Although the effect of eugenol for such cases is less than 8%, a more significant effect can be observed in terms of monolayer expansion since even at higher surface pressures, the isotherm for the mixed monolayer is shifted to higher areas in a percentage higher than 15%. PM–IRRAS spectra shows that the ratio between the area below the curves for bands attributed to the antisymmetric (2916 cm−1) and symmetric (2849 cm−1) stretches is 1.6 for pure DPPS monolayer, and 1.4 for the mixed monolayer (Fig. 4). This indicates a decrease of the order to monolayer related to the lower compressibility. However, the bands for the hydrophilic region do not change considerably in terms of position, showing peaks at 1267 cm−1 (phosphate), 1733 cm−1 (carbonyl) and 1655 cm−1 (water vibrations, being an effect related to the difference of reflectivity of the interface covered and uncovered by the monolayer [23]). However, the relative intensity for the phosphate band increased considerately in relation to the other bands, which indicates that the phosphate groups are affected by the incorporation of eugenol. To this point, these results show that the drug may interact with both lipids, the zwitterionic (DPPC) and the negatively charged (DPPS), affecting the hydrophilic and hydrophobic moieties. However, while for DPPS a significant expansion of the monolayer was observed for the entire range of compression, for DPPC only a slight expansion was observed in intermediate surface pressures, and monolayer condensation is observed at higher surface pressures. Also a more significant decrease of the compressibility modulus is observed for DPPC at
Fig. 3. Surface pressure-area (A) and compressibility modulus-area (B) isotherms for DPPS and DPPS-eugenol (2% in mol) monolayers.
Fig. 4. PM–IRRAS for DPPS and DPPS-eugenol (2% in mol) monolayers.
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higher stages of compression. This fact then suggests that the interaction of eugenol with cell membrane models is modulated by the lipid composition. The polar termination of these lipids (serine and choline) is found in natural membranes [24], including those for bacteria [25]. Although positive charged lipids are not frequently found in cell membranes, we decided to study the interactions of this drug with a synthetic positively charged lipid in order to obtain more information about the role of the charge on the drug-monolayer interaction. In this sense, Fig. 5 shows the π-A isotherms for DODAB, which presents a fluid state and a kink point at approximately 30 mN/m. With eugenol, the monolayer is expanded and a transition is observed at 20–25 mN/m. This transition can be better observed in the panel B, where a decrease in the compressibility modulus is observed at approximately 115 Å2. The fluid state occurs in practically all range of compression for the pure and also for the mixed monolayer. This fact can be attested by the compressibility curve where compressibility modulus values no higher than 80 mN/m are obtained, pointing that the monolayer does not reach the LC state. The surface pressure of collapse in this case is not decreased with drug incorporation as observed for the other lipids. PM–IRRAS (Fig. 6) shows that the spectra are clearly affected when the drug is present. Panel A shows a higher relative contribution for CH3 for DODAB (2871 and 2948 cm− 1) than it was for DPPC and DPPS. For CH2 the antisymmetric/symmetric ratio goes from 1.1 for the pure monolayer to 0.8 for the mixed monolayer, also pointing to
Fig. 6. PM–IRRAS for DODAB and DODAB-eugenol (2% in mol) monolayers.
Fig. 5. Surface pressure-area (A) and compressibility modulus-area (B) isotherms for DODAB and DODAB-eugenol (2% in mol) monolayers.
an expressive change in the order of the monolayer. Furthermore, the positions of the peaks are shifted to higher wavenumbers upon eugenol incorporation. Panel B shows a band centered at 995 cm−1, which is attributed to C–N stretching of the hydrophilic group. This band does not change significantly with the incorporation of the drug. This fact suggests that drug is repelled by the positively charged groups of DODAB and prefers to go deeper into the alkyl chains of the lipid. This differs significantly from DPPS and DPPC where the interaction is detected not only through the alkyl chains but also through the polar groups. This therefore indicates a marked interaction of the drug with the polar groups of the lipid encountered in the membrane. The location of the drug in the lipid membrane is an important issue, being discussed in the literature for liposomes. Reiner et al. [26] suggested that eugenol is able to insert in egg phosphatidylcholines, being located in the region between the polar groups, with the glycerol and first atoms of the acyl chain. This can reduce the repulsive forces among the lipid head groups, permitting closer molecular packing and decreasing the mobility of hydrocarbon chains, as revealed by 1H-NMR. It can be therefore suggested that the nature of lipid influences the action of the drug on the cell membrane models. The large changes arise from the fluidity of the films, affecting the rheological properties of the monolayer. The insertion of the drug then affects the surface relaxation and surface elasticity of the membrane, which could be associated to the ability of a natural membrane to compress and expand reversibly to respond to cell events such as ion transport, cell reproduction and cell signaling. One may speculate that such properties may be related to the possible action of eugenol as a pharmaceutical compound acting in cell membranes.
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4. Conclusions The behavior of eugenol in cell membrane models represented with lipid Langmuir monolayers could be investigated, pointing that the lipid–drug interaction depends remarkably on the chemical nature of the lipid. Eugenol expanded DPPS monolayers in a greater extension than for DPPC monolayers, for which the interaction could be inferred more in terms of the changes in surface compressibility. For DODAB monolayers, also a notable expansion was observed. While for DPPS and DPPC, PM–IRRAS showed that the drug incorporation affects both regions of the lipid, the polar and the alkyl groups, for DODAB this change was more significant for the alkyl groups. These results point therefore that the interaction of eugenol with lipids at the air–water interface is modulated by the lipid monolayer composition. We expect that these results have a substantial impact on the comprehension on the molecular mechanism of action of this compound in biological surfaces. The possible biological implications of these findings may be related to the strong effects caused by incorporating the drug into the cell membrane model, especially at surface pressure values that approximate natural membrane lateral pressures. Acknowledgments This process was supported by FAPESP (2013/10213-1) and CNPq. G.E.G. Gonçalves was a CNPq fellow (119107/2013-9). References [1] S. Ali, R. Prasad, A. Mahmood, I. Routray, T.S. Shinkafi, K. Sahin, & O. Kucuk, J. Cancer Prev. 19 (2014) 288–300. [2] D.F. Cortés-Rojas, C.R. de Souza, & W.P. Oliveira, Asian Pac. J. Trop. Biomed. 4 (2014) 90–96.
[3] S. Chericoni, J.M. Prieto, P. Iacopini, P. Cioni, & I. Morelli, J. Agric. Food Chem. 15 (2005) 4762–4765. [4] G. Singh, S. Maurya, M.P. DeLampasona, & C.A. Catalan, Food Chem. Toxicol. 45 (2007) 1650–1661. [5] J.P. Dai, X.F. Zhao, J. Zeng, Q.Y. Wan, J.C. Yang, W.Z. Li, X.X. Chen, G.F. Wang, & K.S. Li, Plos One 8 (2013) e61026. [6] E. Horvathova, J. Navarova, E. Galova, A. Sevcovicova, L. Chodakova, Z. Snahnicanova, M. Melusova, K. Kozics, & D. Slamenova, J. Agric. Food Chem. 62 (2014) 6632–6639. [7] I. Al-Sharif, A. Remmal, & A. Aboussekhra, Cancer 13 (2013) 600. [8] T. Koh, Y. Murakami, S. Tanaka, M. Machino, H. Onuma, M. Kaneko, M. Sugimoto, T. Soga, M. Tomita, & H. Sakagami, In Vivo 27 (2013) 233–243. [9] S.E. Janes, C.S. Price, & D. Thomas, Eur. J. Pediatr. 164 (2005) 520–522. [10] E. Nagababu, & N. Lakshmaiah, Mol. Cell. Biochem. 166 (1997) 65–71. [11] S. Fujisawa, Y. Kadoma, & Y. Komoda, J. Dent. Res. 67 (1988) 1438–1441. [12] H. Brockman, Curr. Opin. Struct. Biol. 9 (1999) 438–443. [13] L. Caseli, M.E.D. Zaniquelli, R.P.M. Furriel, & F.A. Leone, Colloids Surf. B 30 (2013) 273–282. [14] T.F. Schmidt, F.J. Pavinatto, L. Caseli, M.L.C. Gonzaga, S.A. Soares, N.M.P.S. Ricardo, & O.N. Oliveira, J. Colloid Interface Sci. 330 (2009) 84–89. [15] M.R. Lourenzoni, A.M. Namba, L. Caseli, L. Degreve, & M.E.D. Zaniquelli, J. Phys. Chem. B 111 (2007) 11318–11329. [16] C.P. Pascholati, E.P. Lopera, F.J. Pavinatto, L. Caseli, T.M. Nobre, M.E.D. Zaniquelli, T. Viitala, C. D'Silva, & O.N. Oliveira, Colloids Surf. B 74 (2009) 504–510. [17] T. Goto, & L. Caseli, J. Colloid Interface Sci. 431 (2014) 24–30. [18] J.V.N. Ferreira, S.S. Grecco, J.H.G. Lago, & L. Caseli, Mat. Sci. Eng. C 48 (2015) 112–117. [19] C. Sebaaly, A. Jraij, H. Fessi, C. Charcosset, & H. Greige-Gerges, Food Chem. 178 (2015) 52–62. [20] Z. Yao, W. Namkung, E.A. Ko, J. Park, L. Tradtrantip, & A.S. Verkman, Plos One 7 (2012) e38030–e38036. [21] A. Brume, Biochim. Biophys. Acta 557 (1979) 32–44. [22] C. Huang, J.R. Lapides, & I.W. Levin, J. Am. Chem. Soc. 104 (1982) 5926–5930. [23] D. Blaudez, J.M. Turlut, J. Dufourcq, D. Bard, T. Buffeteau, & B. Desbat, J. Chem. Soc. Faraday Trans. 92 (1996) 525–530. [24] Y. Honma, T. Kasukabe, & M. Hozumi, Biochim. Biophys. Acta 721 (1982) 83–86. [25] M.S. Jurkowitz, A. Patel, L.C. Wu, A. Krautwater, D.R. Pfeiffer, & C.E. Charles, Biochim. Biophys. Acta Biomembr. 1848 (2015) 742–751. [26] G.N. Reiner, L.F. Fracto, E. de Paula, M.A. Perillo, & D.A. García, J. Biomater. Nanobiotechnol. 4 (2013) 28–34.
Contents lists available at ScienceDirect
Biophysical Chemistry journal homepage: http://www.elsevier.com/locate/biophyschem
The interaction of eugenol with cell membrane models at the air–water interface is modulated by the lipid monolayer composition Giulia E.G. Gonçalves, Fernanda S. de Souza, João Henrique G. Lago, Luciano Caseli ⁎ Institute of Environmental, Chemical, and Pharmaceutical Sciences, Federal University of São Paulo (UNIFESP), Diadema, São Paulo, Brazil
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Langmuir monolayers of lipids are formed at the air–water interface. • Eugenol is incorporated to the monolayer. • Interactions at the molecular level are identified with tensiometry and PM–IRRAS. • Intermolecular interactions are modulated by the monolayer lipid composition.
a r t i c l e
i n f o
Article history: Received 3 July 2015 Received in revised form 21 July 2015 Accepted 26 July 2015 Available online 29 July 2015 Keywords: Eugenol Langmuir monolayer Air–water interface Biomembranes Drug
a b s t r a c t Eugenol, a natural phenylpropanoid derivative with possible action in biological surfaces as microbicide, anesthetic and antioxidant, was incorporated in lipid monolayers of selected lipids at the air–water interface, representing cell membrane models. Interaction of eugenol with the lipids dipalmitoylphosphatidylcholine (DPPC), dioctadecyldimethylammonium bromide (DODAB), and dipalmitoylphosphatidylserine (DPPS) could be inferred by means of surface pressure-area isotherms and Polarization–Modulation Reflection–Absorption Spectroscopy. The interaction showed different effects on the different lipids. A higher monolayer expansion was observed for DPPS and DODAB, while more significant effects on the polar groups of the lipids were observed for DPPS and DPPC. These results pointed to the fact that the interaction of eugenol with lipid monolayers at the air–water interface is modulated by the lipid composition, which may be important to comprehend at the molecular level the interaction of this drug with biological surfaces. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Eugenol is an allylphenol, belonging to the class of natural products known as phenylpropanoids. It is extracted from essential oils such as, ⁎ Corresponding author at: Rua São Nicolau, 210, Unifesp, Diadema São Paulo, 09913-030, Brazil. E-mail address: [email protected] (L. Caseli).
http://dx.doi.org/10.1016/j.bpc.2015.07.007 0301-4622/© 2015 Elsevier B.V. All rights reserved.
nutmeg, clove oil, cinnamon, basil and bay leaf [1–3]. This compound has been reported to be used in flavorings, perfumeries, and in medicine as local antiseptics and anesthetics. Also it is considered as a bactericide, and an antiviral compound [4, 5], and can be employed as restorative applications in dentistry, in antioxidants for plastics and rubbers, as an anesthetic, and in some mousetraps. Also it has been reported that eugenol kills certain human colon
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cancer cell lines in vitro and in vivo [6–8]. Furthermore, it is reported that it is hepatotoxic, causing damage to living beings [9]. In this sense, it is of interest to study the interactions of this compound in biointerfaces, such as cell membranes. The understanding of how eugenol interacts with these surfaces may help comprehend its molecular mechanism of action in cell membranes. It is reported that eugenol inserted in liposomes inhibits the enzymatic action of xanthine oxidase–xanthine-iron [10]. Also, it has been shown that eugenol prevents membrane damaging events for which its presence resulted in the inhibition for the formation of malondialdehyde in irradiated liposomes. Fujisawa et al. [11] reported that the cytotoxic activity of eugenol against cells was enhanced by visible-light irradiation due to its high redox potential. A proper system to be used as a model for cell membranes is the Langmuir monolayers, which is composed by monomolecular films of amphiphilic compounds at the liquid–gas interface. When organic solutions of membrane lipids are spread on the air–water interface, a model for half a membrane is formed [12], and interactions with proteins [13], polysaccharides [14], peptides [15] and drugs [16] can be investigated by means of tensiometry, vibrational spectroscopy and other surface specific techniques. Particularly, the interaction of several drugs with lipids at the air–water interface have been studied in Langmuir monolayers of lipids [16–18], and to the best of our knowledge no report on the interaction of eugenol with cell membrane models represented by a Langmuir monolayer has been reported. However, some studies have been shown the insertion of eugenol in bilayers [10–19], which makes promising the study with Langmuir monolayers as a complementary model. Also, these monomolecular films are useful to understand molecular interactions for other kind of lipid-drug systems, such as in liposomes in drug delivery systems. In this present work, we studied the interaction of eugenol with Langmuir monolayers composed of selected lipids. In order to better understand the role of the chemical nature of the lipid in these interactions, three different lipids were employed, a zwitterionic one, dipalmitoylphosphatidylcholine (DPPC), a positively charged one, dioctadecyldimethylammonium bromide (DODAB), and a negatively charged lipid, dipalmitoylphosphatidylserine (DPPS). 2. Materials and methods 2.1. General DPPC, DODAB and DPPS were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in chloroform (Synth, Diadema, Brazil) to a concentration of 0.5 mg/mL. The monolayer subphase approximated physiological conditions and consisted of a 50 mM phosphate buffer and 150 mM NaCl at a pH of 7.4. The water employed was purified using a MilliQ-Plus System (resistivity 18.2 MΩ cm, pH 5.5). LREIMS was measured in MS-QP-5050A mass spectrometer, operating at electron impact (70 eV). 1H and 13C NMR spectra were recorded, respectively, at 300 and 75 MHz in a Bruker Advance II 300 spectrometer using CDCl3 (TediaBrazil) as solvent and TMS (tetramethylsilane) as internal standard. Chemical shifts (δ) are reported in ppm and coupling content (J) in Hz. 2.2. Isolation of eugenol Aiming the isolation of eugenol, twigs of Nectranda leucantha were collected in the municipality of Cubatão, São Paulo State, Brazil in November 2013. The dried and powdered plant material (300 g) was exhaustively extracted with n-hexane, and its concentration was reduced under pressure, obtaining about 9 g of n-hexane extract. Part of this crude material (7 g) was subjected to column chromatography over silica gel using increasing amounts of EtOAc in n-hexane to afford six fractions (A to F). Fraction D (76 mg) was purified by Sephadex LH-20 using MeOH as eluent to afford 13 mg of pure eugenol.
Eugenol was characterized by NMR (300 and 75 MHz, CDCl3) and follows: δH 6.67 (d, J = 2.0 Hz, H-2), 6.84 (d, J = 8.1 Hz, H-5), 6.70 (dd, J = 8.1 and 2.0 Hz, H-6), 3.32 (d, J = 6.6 Hz, H-7), 5.93 (m, H-8), 5.06 (m, H-9), 3.87 (s, 3-OCH3). δC 131.9 (C-1), 111.1 (C-2), 146.5 (C3), 143.9 (C-4), 121.2 (C-5), 114.3 (C-6), 39.9 (C-7), 137.8 (C-8), 115.5 (C-9), 55.9 (3-OCH3). LREIMS m/z (rel. int.) 164 (M+, 100), 149 (47), 137 (31), 131 (40), 121 (20), 103 (25), 91 (20), 77 (21). 2.3. Preparation of monolayers The Langmuir monolayers were obtained by spreading a chloroform solution of DPPC, DPPS or DODAB on the surface of an aqueous buffer solution. For preliminary tests, eugenol solutions, dissolved in chloroform to a concentration of 0.5 mg/mL, were also spread alone at the air–water interface in order to test the surface activity of this compound. For mixed eugenol-lipid monolayers, first the lipid was spread on the air– water interface. Then, 20 min was allowed for chloroform evaporation, and aliquots of eugenol to render a 2% in mol-lipid were carefully injected in the aqueous subphase. Other proportions were essayed and this value represents a limit for the effect of this drug in terms of expansion of the monolayer. For higher amounts the isotherms do not present reproducibility probably because effects related to the formation of aggregates. Also, considering that drugs interacting with membranes are inserted in relative small amounts, this proportion must approximate conditions in vivo. Surface pressure-area (π-A) isotherms were obtained in a mini-KSV Langmuir trough equipped with a surface pressure sensor (Wilhelmy method), with an interface compression rate of 5 Å2 molecule−1 min−1. After allowing 20 min for evaporation of chloroform, the monolayer was compressed until the collapse is reached. Each isotherm shown in this paper was repeated at least three times for checking the reproducibility, and no variations higher than 0.1 mN/m was allowed. Then for each graph, a representative isotherm is shown. For PM–IRRAS studies, the monolayer was compressed until the desired surface pressure (30 mN/m). The surface pressure was maintained at the desired surface pressure by moving the barriers, and the stabilization of the monolayer was monitored until no additional movement of the barriers was needed. The PM–IRRAS measurements were then taken using a KSV PMI 550 instrument (KSV Instruments, Ltd., Helsinki, Finland) at a fixed incidence angle of 80°. Each spectrum shown in this paper was repeated at least three times for checking the reproducibility and a representative spectrum is shown. All experiments were carried out at a controlled room temperature (25 °C). 3. Results and discussion After isolation of eugenol from twigs of N. leucantha, its structure was defined by analysis with 1H, 13C NMR and LREIMS data and compared with data previously described in the literature [11,20]. Eugenol spread alone on the air–water interface in the concentration used in this work does not present any surface activity, i.e. its spreading on the air–water interface does not decrease the surface tension of water. When the interface is compressed, thus increasing the surface molecular density, the surface pressure does not raise more than 1 mN/m, which confirms that this compound does not form Langmuir monolayers. As they are insoluble in water and cannot leave the interface, this fact can be attributed to the low spreading coefficient of eugenol, which must cause its aggregation at the air–water interface. When incorporated in lipid monolayers, the surface activity of eugenol is detected, as inferred by means of analysis of the surface pressurearea isotherms. Fig. 1A shows the action of eugenol in Langmuir monolayers of DPPC. This lipid presents a typical π-A isotherm [21], with a plateau at approximately 14 mN/m, representing the transition between the states liquid-expanded (LE) and liquid-condensed (LC). The monolayer collapses at 55–60 mN/m in molecular areas of 45–50 Å2.
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indicates that the monolayer is less elastic to the compression, which may be associated to the ability of the monolayer to re-arrange molecularly upon the effect of lateral compression. It is important to mention that other proportions were essayed and the proportion in mol of 2% of eugenol in relation to the lipids represented a limit for the effect of this drug in terms of expansion of the monolayer. For higher amounts, the isotherms do not present reproducibility, probably owing to supramolecular effects such as the formation of aggregates. Lower amounts present similar effects until 0.5%, and below this value no significant effect was observed in the isotherms. Also, an evaluation of the possible loss of area monolayer was tested upon repetitive compression–expansion cycles. This was carried out until 5 cycles for all monolayers, pure or mixed, and no relevant hysteresis was observed. Fig. 2 shows the PM–IRRAS spectra for the monolayers at 30 mN/m. This value was chosen because it is related to the value of lateral pressure encountered in natural membranes [21]. Panel A shows the bands assigned for CH2 stretches. The ratio between the area below the curves for bands attributed to the antisymmetric (2915 cm−1) and symmetric (2848 cm−1) stretches is related to an order parameter [22]. For pure DPPC this value is 1.9; and for eugenol-DPPC it is 1.4. This decrease of order is closely related to the lower compressibility, indicating the effect of the drug. Also the band centered at 2878 cm−1, attributed to CH3 groups, is more evident for the mixed monolayer, whereas for the pure DPPC monolayer, it seems that this band appears as a shoulder.
Fig. 1. Surface pressure-area (A) and compressibility modulus-area (B) isotherms for DPPC and DPPC-eugenol (2% in mol) monolayers.
When mixed with eugenol, apparently no change is observed in the gaseous and liquid-expanded region (from 145 to 75 Å2). Further compression leads to a shift of the isotherm to higher areas in relation to the isotherm for pure DPPC. This fact is observed until the surface pressure of 32 mN/m. For higher surface pressures, the isotherm for the mixed film is shifted to lower molecular areas and collapse is observed at 50 mN/m. The shift to higher molecular area indicates the expansion of the lipid monolayer and can be associated to the incorporation of the drug inside the alkyl chains of the phospholipids. This fact may also change the transition of phases for DPPC since a transition typical for a pure substance (a plateau in the isotherm) is not well defined anymore. The shift of the isotherm to lower molecular areas indicates the condensation of the monolayer and may indicate either expulsion of the drug from the monolayer or molecular accommodation of the drug during the compression. As the surface pressure of collapse and the monolayer compressibility changed at such high surface pressures, it is likely that the second hypothesis is the most probable. It is important to emphasize that for these specific curves, the isotherms were repeated 5 times and no significant deviation (i.e. more than 0.1 mN/m) was observed, assuring the reproducibility of the experiments. Also, it is clear that the binary mixtures formed are not ideal, and then some changes in the isotherms at high surface pressures may be a consequence of the partial squeezing of eugenol from the monolayer or the self-segregation into enriched eugenol microdomains. Fig. 1B shows the compressibility modulus is lower for the mixed monolayer in relation to the pure lipid monolayer. This not only confirms the incorporation of eugenol at such high values of surface pressure, but also
Fig. 2. PM–IRRAS for DPPC and DPPC-eugenol (2% in mol) monolayers at 30 mN/m.
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Panel B for Fig. 2 shows the region for hydrophilic groups of DPPC. The band centered at 1723 cm−1 is attributed to carbonyl stretches, and is shifted to 1736 cm−1 upon incorporation of eugenol. The band centered at 1225 cm− 1 is attributed to phosphate stretches and is shifted to 1261 cm−1 owing to eugenol incorporation. The band centered in 1168 cm−1 may be attributed to ring vibrations of the drug. These results show that the drug may affect also the hydrophilic groups of DPPC. Fig. 3 shows the surface pressure-area isotherms for DPPS. With the presence of eugenol the isotherm is shifted to higher areas. This effect is more prominent than that observed for DPPC, and indicated noticeably the expansion of the monolayer, probably caused by the penetration of eugenol in the monolayer and to repulsions due to its presence. Eugenol is not charged in this pH, but DPPS is, so these repulsions must have origin from dipole–charge interactions. Due to its expansion, even the initial surface pressure begins in values above zero (about 10 mN/m). Also the rheological properties are affected since Fig. 3B shows significant changes in the compressibility of the monolayer. The LE–LC transition occurs for pure DPPS between the areas of 90 and 60 Å2 at 15–16 mN/m. With the presence of eugenol, this range occurs between 120 and 90 Å2 at 16–17 mN/m. These higher values of area are an effect of the monolayer expansion. For the mixed monolayer, another transition is observed between 80 and 70 Å2 at the surface pressure of 25 mN/m, and may be attributed to molecular accommodations of the drug embedded in the lipid monolayer during the compression. The maximum value of compressibility modulus (480 mN/m) is lower than for the pure lipid
monolayer (520 mN/m), and the collapse pressure decreases from 70 to 66 mN/m. Although the effect of eugenol for such cases is less than 8%, a more significant effect can be observed in terms of monolayer expansion since even at higher surface pressures, the isotherm for the mixed monolayer is shifted to higher areas in a percentage higher than 15%. PM–IRRAS spectra shows that the ratio between the area below the curves for bands attributed to the antisymmetric (2916 cm−1) and symmetric (2849 cm−1) stretches is 1.6 for pure DPPS monolayer, and 1.4 for the mixed monolayer (Fig. 4). This indicates a decrease of the order to monolayer related to the lower compressibility. However, the bands for the hydrophilic region do not change considerably in terms of position, showing peaks at 1267 cm−1 (phosphate), 1733 cm−1 (carbonyl) and 1655 cm−1 (water vibrations, being an effect related to the difference of reflectivity of the interface covered and uncovered by the monolayer [23]). However, the relative intensity for the phosphate band increased considerately in relation to the other bands, which indicates that the phosphate groups are affected by the incorporation of eugenol. To this point, these results show that the drug may interact with both lipids, the zwitterionic (DPPC) and the negatively charged (DPPS), affecting the hydrophilic and hydrophobic moieties. However, while for DPPS a significant expansion of the monolayer was observed for the entire range of compression, for DPPC only a slight expansion was observed in intermediate surface pressures, and monolayer condensation is observed at higher surface pressures. Also a more significant decrease of the compressibility modulus is observed for DPPC at
Fig. 3. Surface pressure-area (A) and compressibility modulus-area (B) isotherms for DPPS and DPPS-eugenol (2% in mol) monolayers.
Fig. 4. PM–IRRAS for DPPS and DPPS-eugenol (2% in mol) monolayers.
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higher stages of compression. This fact then suggests that the interaction of eugenol with cell membrane models is modulated by the lipid composition. The polar termination of these lipids (serine and choline) is found in natural membranes [24], including those for bacteria [25]. Although positive charged lipids are not frequently found in cell membranes, we decided to study the interactions of this drug with a synthetic positively charged lipid in order to obtain more information about the role of the charge on the drug-monolayer interaction. In this sense, Fig. 5 shows the π-A isotherms for DODAB, which presents a fluid state and a kink point at approximately 30 mN/m. With eugenol, the monolayer is expanded and a transition is observed at 20–25 mN/m. This transition can be better observed in the panel B, where a decrease in the compressibility modulus is observed at approximately 115 Å2. The fluid state occurs in practically all range of compression for the pure and also for the mixed monolayer. This fact can be attested by the compressibility curve where compressibility modulus values no higher than 80 mN/m are obtained, pointing that the monolayer does not reach the LC state. The surface pressure of collapse in this case is not decreased with drug incorporation as observed for the other lipids. PM–IRRAS (Fig. 6) shows that the spectra are clearly affected when the drug is present. Panel A shows a higher relative contribution for CH3 for DODAB (2871 and 2948 cm− 1) than it was for DPPC and DPPS. For CH2 the antisymmetric/symmetric ratio goes from 1.1 for the pure monolayer to 0.8 for the mixed monolayer, also pointing to
Fig. 6. PM–IRRAS for DODAB and DODAB-eugenol (2% in mol) monolayers.
Fig. 5. Surface pressure-area (A) and compressibility modulus-area (B) isotherms for DODAB and DODAB-eugenol (2% in mol) monolayers.
an expressive change in the order of the monolayer. Furthermore, the positions of the peaks are shifted to higher wavenumbers upon eugenol incorporation. Panel B shows a band centered at 995 cm−1, which is attributed to C–N stretching of the hydrophilic group. This band does not change significantly with the incorporation of the drug. This fact suggests that drug is repelled by the positively charged groups of DODAB and prefers to go deeper into the alkyl chains of the lipid. This differs significantly from DPPS and DPPC where the interaction is detected not only through the alkyl chains but also through the polar groups. This therefore indicates a marked interaction of the drug with the polar groups of the lipid encountered in the membrane. The location of the drug in the lipid membrane is an important issue, being discussed in the literature for liposomes. Reiner et al. [26] suggested that eugenol is able to insert in egg phosphatidylcholines, being located in the region between the polar groups, with the glycerol and first atoms of the acyl chain. This can reduce the repulsive forces among the lipid head groups, permitting closer molecular packing and decreasing the mobility of hydrocarbon chains, as revealed by 1H-NMR. It can be therefore suggested that the nature of lipid influences the action of the drug on the cell membrane models. The large changes arise from the fluidity of the films, affecting the rheological properties of the monolayer. The insertion of the drug then affects the surface relaxation and surface elasticity of the membrane, which could be associated to the ability of a natural membrane to compress and expand reversibly to respond to cell events such as ion transport, cell reproduction and cell signaling. One may speculate that such properties may be related to the possible action of eugenol as a pharmaceutical compound acting in cell membranes.
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4. Conclusions The behavior of eugenol in cell membrane models represented with lipid Langmuir monolayers could be investigated, pointing that the lipid–drug interaction depends remarkably on the chemical nature of the lipid. Eugenol expanded DPPS monolayers in a greater extension than for DPPC monolayers, for which the interaction could be inferred more in terms of the changes in surface compressibility. For DODAB monolayers, also a notable expansion was observed. While for DPPS and DPPC, PM–IRRAS showed that the drug incorporation affects both regions of the lipid, the polar and the alkyl groups, for DODAB this change was more significant for the alkyl groups. These results point therefore that the interaction of eugenol with lipids at the air–water interface is modulated by the lipid monolayer composition. We expect that these results have a substantial impact on the comprehension on the molecular mechanism of action of this compound in biological surfaces. The possible biological implications of these findings may be related to the strong effects caused by incorporating the drug into the cell membrane model, especially at surface pressure values that approximate natural membrane lateral pressures. Acknowledgments This process was supported by FAPESP (2013/10213-1) and CNPq. G.E.G. Gonçalves was a CNPq fellow (119107/2013-9). References [1] S. Ali, R. Prasad, A. Mahmood, I. Routray, T.S. Shinkafi, K. Sahin, & O. Kucuk, J. Cancer Prev. 19 (2014) 288–300. [2] D.F. Cortés-Rojas, C.R. de Souza, & W.P. Oliveira, Asian Pac. J. Trop. Biomed. 4 (2014) 90–96.
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