Use of Ionic Liquids-like Surfactants for the Generation of Unilamellar Vesicles with Potential Applications in Biomedicine

1 Use of ionic liquids-like surfactants for the generation of unilamellar vesicles with potential applications in biomedicine Cristian M. O. Lépori*[a], N. Mariano Correa[b,c], Juana J. Silber[b,c], R. Darío Falcone[b,c], Manuel López-López[d] and M. Luisa Moyá[e] [a] Dr. C. M. O. Lépori. Instituto de Física Enrique Gaviola (IFEG), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) – Universidad Nacional de Córdoba (UNC), Medina Allende s/n, Ciudad Universitaria, X5016LAE, Córdoba, Argentina. [b] Dr. N. M. Correa, Prof. J. J. Silber and Dr. R. D. Falcone. Departamento de Química, Universidad Nacional de Río Cuarto (UNRC), Agencia Postal # 3, C.P. X5804BYA, Río Cuarto, Argentina. [c] Instituto para el Desarrollo Agroindustrial y de la Salud (IDAS), CONICET – UNRC. Agencia Postal # 3, C.P. X5804BYA, Río Cuarto, Argentina. [d] Dr. M. López-López. Departamento de Ingeniería Química, Química Física y Ciencias de Materiales, Centro de Ciencia y Tecnología, Universidad de Huelva, Campus ‘El Carmen’, Facultad de Ciencias Experimentales, E-21071, Spain. [e] Prof. M. L. Moyá. Departamento de Química Física, Universidad de Sevilla, c/ Prof. García González 1, 41012 Sevilla, Spain. * Corresponding-Author: Dr. Cristian M. O. Lépori. E-mail: [email protected] Page 1 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 ABSTRACT The goal of this work is to understand the influence of the counterion nature on the organized systems formed by 1,4-bis-2-ethylhexylsulfosuccinate surfactants in aqueous solutions and, how these aggregates will have influence on the DNA- surfactants interactions. With this in mind, two ionic liquid-like surfactants were investigated: 1-butyl-3-methylimidazolium 1,4-bis-2-ethylhexylsulfosuccinate (bmim- AOT) and 1-hexyl-3-methylimidazolium 1,4-bis-2-ethylhexylsulfosuccinate (hmim- AOT). Measurements of dynamic light scattering (DLS), zeta potential, TEM images, fluorescence and UV-Visible spectroscopy were performed in order to study the characteristics of the vesicles formed by bmim-AOT and hmim-AOT. Regarding the determination of the interaction of the surfactants with DNA, circular dichroism was used. The results obtained showed that bmim-AOT and hmim-AOT ionic liquid-like surfactants spontaneously form unilamellar vesicles in water at very low surfactant concentrations. The characteristics of these aggregates are dependent on the length of the tail of the counterions. The length of the hydrophobic chains of the counterions also influences on the DNA:surfactant interactions through hydrophobic effects. KEYWORDS: ionic liquid-like surfactant, vesicles, bmim-AOT, hmim-AOT, DNA. Page 2 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 INTRODUCTION Ionic liquids (ILs) are a friendly class of compounds which have received significant attention as alternatives to conventional organic solvents.1–3 The great impact that ILs have in chemistry is predominantly due to their properties that can be adapted in the synthesis procedure by a large variety of cation-anion combinations.4 An attractive field of research deals with the synthesis of amphiphilic ILs.5–7 These new materials, called IL-like surfactants, have been used to create a different kind of organized systems such as direct micelles, reverse micelles and vesicles.3,5–11 Vesicles are spherical organized systems formulated in water by some amphiphilic compounds (usually phospholipids). In these aggregates, the nonpolar bilayer surrounds an aqueous core.12 Particularly, large unilamellar vesicles (LUVs) can be prepared using different techniques (such as ultra-sonication or extrusion) in order to convert multilamellar vesicles (formed spontaneously) into LUVs.12,13 These vesicles can enhance drug stability, therapeutic effects, and uptake of the solubilized drug into the target site with reduced toxicity.12 These organized systems can also be used for the development of new techniques for deoxyribonucleic acid (DNA) extraction, gene therapy and gene transfection.14,15 However, it is mandatory to know which are the DNA-surfactant interactions present and, how they can modify, or not, the DNA structure. It is well-established knowledge that highly charged counterions have an impact on DNA compaction.16,17 Therefore, the ILs can influence the DNA structure: the cations mostly interact with the DNA phosphate groups through electrostatic attractions, whereas the anions interact with the nucleobases.18 For these reasons, the design of new IL-like surfactants that form organized systems in aqueous solution, with the ability to solubilize hydrophobic molecules, and that can also interact with DNA is a very interesting area to investigate. Recently10 we reported the synthesis of the IL-like Page 3 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 4 surfactant 1-butyl-3-methylimidazolium 1,4-bis-2-ethylhexylsulfosuccinate (bmim- AOT, Scheme 1), resulting from the mixture of sodium 1,4-bis-2- ethylhexylsulfosuccinate (Na-AOT) and 1-butyl-3-methylimidazolium chloride (bmim- Cl). This IL-like surfactant was isolated from the reactive mixture (removing the Na+ and Clcounterions) and showed properties absolutely different from Na-AOT.10 Particularly, we studied the physicochemical properties of reverse micelles in nonpolar solvents formed by bmim-AOT. It must be noted that, the properties of this surfactant in aqueous media were reported previously19,20 but, it is not clear which kind of aggregates are form in water. Moreover, the very diluted concentration regime, where the aggregates-aggregates interactions are minimized, was not investigated. Thus, the kind of aggregates that bmim-AOT forms in water is not revealed until nowadays. In the present work, we want to investigate the ability of this surfactant to form aggregates in aqueous solution, working in quite diluted solutions. Thus, we will show that, at bmim-AOT concentration lower than 1.5x10-3 M, unilamellar vesicles are formed. Futhermore, in order to investigate the effect that the counterions have on the properties of the formed aggregates, 1-hexyl-3-methylimidazolium 1,4-bis-2- ethylhexylsulfosuccinate (hmim-AOT, Scheme 1) was also synthesized. The study of this system will provide information about the influence of the counterion structure on these novel organized systems and, how may influence in their possible applications. In this way, physicochemical properties such as size, surface charge, micropolarity and microviscosity of the unilamellar vesicles were also investigated. Particularly, because we want to use these vesicles as a drug delivery agent21 or for transfection of DNA molecules.22 bmim-AOT and hmim-AOT vesicles were investigated using dynamic light scattering (DLS), zeta potential and transmission electron microscopy (TEM) techniques. Besides, the use of two different fluorescent molecular probes, 2,2'-[[4-[(4- Page 4 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 5 nitrophenyl)azo]phenyl]imino]bisethanol23–25 (disperse red 19, DR9, Scheme 1) and 1,3-dipyrenylpropane,26,27 (P3P, Scheme 1), which are incorporated into the vesicles, provided information about the polarity and the viscosity of their microenvironment.28,29 Finally, the interactions between bmim-AOT and hmim-AOT vesicles and calf thymus DNA were studied using circular dichroism (CD).30–34 bmim-AOT N N OO S O O O O -O hmim-AOT N N OO S O O O O -O NN N O2N DR19 HO OH NN P3P Scheme 1. Molecular structures of the surfactants bmim-AOT and hmim-AOT, and the Page 5 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 6 molecular probes DR19 and P3P. EXPERIMENTAL SECTION Materials 1-hexyl-3-methylimidazolium chloride (hmim-Cl), bmim-Cl, Na-AOT, dichloromethane (DCM) and DR19 were from Sigma-Aldrich (> 99% purity). These compounds were kept under vacuum. P3P was purchased from Molecular Probes, Inc. and used as received. The IL-like surfactant used, bmim-AOT, was obtained according the experimental procedure described in reference.10 Hmim-AOT was prepared following the same methodology,10 using as precursors hmim-Cl and Na-AOT (Scheme S1). The IL hmim-AOT obtained was a colorless and highly viscous liquid. The formation of hmim-AOT was confirmed by 1H NMR technique. Figure S1 show that all protons corresponding to the anionic (AOT) and to the cationic (hmim+) components are present. The chemical shifts values of the most relevant H of hmim-AOT obtained in CDCl3 are included in Table S1. The NMR spectra for all compounds were performed in CITIUS (Research General Service for the University of Seville), using a Bruker Avance III 500 MHz spectrometer (500.2 MHz for 1H). Also, FT-IR spectrum of hmim- AOT in CCl4 was performed and compared with Na-AOT (Figure S2), observing the presence of the C-H stretching bands of the imidazolium ring. To obtain the FT-IR spectra a Nicolet IMPACT400 FT-IR spectrometer and IR cell of the type Irtran-2 (0.015 mm of path length) from Wilmad Glass (Buena, NJ) were used. The vesicle solutions were prepared by mass and volumetric dilution, from a stock solution of surfactants prepared in deionized distilled water Super Q Millipore Page 6 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 7 (resistivity > 18 MΩ cm), and these samples were used directly in the different experiments without any other procedure, as it was made in other works.6–8 Calf thymus DNA (ctDNA, 99% purity) was purchased from Sigma-Aldrich. The ctDNA concentration (given by phosphate groups) was estimated spectrophotometrically at 260 nm (molar absorptivity of 6600 M-1 cm-1).35 Hydrodynamic diameter and zeta potential measurements A Zetasizer Nano ZS Malvern Instrument Ltd. was used for the measurement of hydrodynamic diameter and the zeta potential at 25 °C. The scattering angle used was 173°. CONTIN was used as the algorithm to obtain the hydrodynamic diameter values. All the experiments were carried out at several different surfactant concentrations however, systems with [surfactant] larger than 7x10-4 M were not prepared to the DLS measurements due to opalescence of the final solutions. At least six hydrodynamic diameters and zeta potentials were measured at each surfactant concentration and the average value (standard deviation) was considered. Transmission Electron Microcospy (TEM) For the TEM experiments, the micrographs were obtained with a JEOL 1200 EXII transmission electron microscope at a working voltage of 80 kV. The TEM samples were prepared by the negative-staining method. Phosphotungstic acid solution (2%) was used as the staining agent.36 Solubilization capacity and micropolarity of vesicle bilayer Taking into account that DR19 is an azo dye insoluble in water, it was used to evaluate the solubilization capacity of the bmim-AOT and hmim-AOT vesicles. Thus, Page 7 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 8 an excess amount of DR19 in surfactant aqueous solutions was left stirring for two hours, at several surfactant concentrations. The resultant solution was filtered, using a standard gravity filtration method, and the dye concentration was determined spectrophotometrically at 495 nm and  = 23000 M-1 cm-1.23,24,28 The absorbance of the dye solutions was measured in a Hitachi UV-3900 spectrophotometer. The temperature was maintained at 25.0 ± 0.1 °C by using a water-jacketed cell compartment. Determination of the microviscosity of vesicle bilayer The vesicles containing P3P (2x10-6 M) solutions were prepared following the method in reference.26 The fluorescence emission spectra of P3P solutions were registered between 350 and 550 nm with an exc = 346 nm. The intensities of the monomer emission (IM) and the excimer emission (IE) were recorded at the wavelength corresponding to the first vibronic peak of the monomer, located near 378 nm, and that of the excimer at around 490 nm, respectively.26 Fluorescence measurements were done in a Hitachi F-2500 fluorescence spectrophotometer. The value of IM/IE ratio reported to each concentration of surfactant was the average of ten spectra. The temperature was kept at 25°C by a water flow cryostat connected to the cell compartment. Circular dichroism Circular dichroism (CD) experiments were performed in a Biologic Mos-450 spectropolarimeter. A circulation thermostatic bath kept the sample temperature at 25.0  0.1 °C. A cuvette of 0.1 cm path length and a scan speed of 50 nm min-1 were used. Prior to use argon was passed through the solutions for more than two hours. The reported spectra were the average of ten runs with a 5 min equilibration before each scan. Page 8 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 9 RESULTS AND DISCUSSION The solubility of bmim-AOT and hmim-AOT in water was investigated. Both ILs were soluble in water up to the evaluated concentration of 1 M, forming opalescent solutions stable over time. This type of opalescent solutions can be taken as indicative of the formation of aggregates of a considerable size since they show a great capacity to scatter the light.37 Different solutions of bmim-AOT and hmim-AOT in water were prepared and the samples were analyzed by DLS. Table 1 shows the apparent hydrodynamic diameter, dapp, values obtained for bmim-AOT and hmim-AOT in water at 25 ºC. Also in Figure S3, the DLS intensity plot for both surfactant in water at [surfactant] = 3x10-4 M is depicted. The DLS data show the formation of aggregates with diameters ranging from 146 - 164 nm for bmim-AOT and, from 93 - 120 nm for hmim-AOT, with low polydispersity indexes (within the range 0.2 - 0.4) in both cases. Moreover, in the range of 2x10-5 M - 7x10-4 M the sizes were independent of the surfactant concentration. The size ranges found for the two surfactants could suggest the formation of vesicles.38 The independency observed on the diameter values measured at different [surfactant] and, particularly the detection of aggregates even at low [surfactant] are good evidences that the aggregates formed (spontaneously) are vesicles and not direct micelles.19,20,39–43 As it is well known the lack of critical aggregation concentration is a peculiarity that distinguishes vesicles13 from direct micelles.38 Moreover, the aggregates obtained at different [surfactant] concentrations do not present high polydispersity indexes, suggesting that they are unilamellar vesicles.6,7 Table 1. Apparent hydrodynamic diameters (dapp) and polydispersity indexes (PDI) of Page 9 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 16 vesicles. This smaller size could be the result of the hmim+ hexyl tails being disposed in a more compact conformation in the vesicle bilayer than the bmim+ butyl tails in the bmim-AOT aggregates. This behavior is similar to the observed for the catanionic IL- like surfactant benzyl-n-hexadecyldimethylammonium 1,4-bis-2- ethylhexylsulfosuccinate used to create vesicles which have a more compact bilayer with greater capacity to solubilize dyes than the DOPC vesicles.8 It is important to bear in mind that the size of the vesicles and the compactness of the bilayer depend, among many other variables, on the effective packaging parameter of the surfactant p, defined as p = v/alc, where v and lc are the volume and the length of the hydrocarbon chain, respectively, and a is the area of the surfactant head group.51 The p value of the vesicles is within the range 1/2 < p < 1, for direct micelles p < 1/3 and for bilayers p ≈ 1. The size of the vesicles is larger when the p value is closer to 1. Therefore, all the factors that increase the v value, increase the packing parameter that affects the size of the vesicles.52 At the surfactant concentrations used in this work, the Na-AOT precursor in water forms mainly direct micelles,45,53,54 however, the results obtained in this work indicate that the two IL-like surfactants prepared form spontaneously vesicles in water. This can be explained as follows: the ionic nature of the polar head group of the precursors (Na-AOT, bmim-Cl and hmim-Cl) and, the need to act as counterions to each other in the new ILs-like surfactants produces changes in the p parameter compared to the Na-AOT precursor. Our hypothesis is that due to the replacement of the Na+ counterion of AOT by bmim+ and hmim+, an increase in the effective area can be expected. However, the bmim+ and hmim+ counterions have a hydrocarbon tail which could be located close to the hydrocarbon tails of AOT, favoring hydrophobic interactions and significantly modifying the effective volume. In this way the v/a ratio would increase, causing an increment in the p value, which could go from p Page 16 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 17 < 1/3 (corresponding to Na-AOT) to p between 1/2 and 1 (corresponding to bmim-AOT and hmim-AOT vesicles). In regard to the zeta potential values, they seem to indicate that there is a somewhat higher presence of hmim+ than bmim+ in the bilayer of the vesicles. This could result in a decrease in the electrostatic repulsions between the negative charges of the AOT, making the v/a ratio slightly lower in hmim-AOT vesicles than in those of bmim-AOT and, therefore, generating a more compact bilayer and smaller vesicles. A possible distribution of the ions at the bilayer in both vesicles is proposed in Scheme 2. O OO S O O O O- O O O S O O O -O O O O S O O O -O O H H O H H O H H O H H O OO S O O O O- O H H O H H O H H O H H O H H N N NN N N N N O H H O H H O O O S O O O O- O H HO H H O H H Scheme 2. Schematic representation of the vesicle interfaces of IL-like surfactants (A: water/bmim-AOT and B: water/hmim-AOT). O OO S O O O O- O O O S O O O O- O O O S OO O -O O H H O H H O H H O H H O OO S O O O O- O H H O H H O H H O H H N N N N N N O H H O H H O O O S O O O O- O H HO H H N N N N O H H A B Page 17 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 18 Once the vesicles systems were investigated, it would be very interesting to explore the possible DNA solubilization in the bilayer and, to investigate the DNA- surfactant interactions. CD experiments were performed in order to investigate the interactions between DNA and the vesicles. Vesicular solutions of bmim-AOT and hmim-AOT were prepared at different surfactant concentrations and, the same amount of ctDNA ([ctDNA] = 5x10-5 M) was added to each of them. Using the CD technique, the characteristic bands of DNA between 220 - 310 nm were analyzed and plotted in Figure 4. Double stranded DNA shows a positive band at  = 278 nm and a negative band at  = 246 nm, corresponding to the stacking of - bases and the helical structure, respectively.55 CD spectra of solutions containing ctDNA with bmim-Cl and hmim-Cl were also registered, in order to know if the cationic precursors of both surfactants interact with the DNA (See Figure S4 in supporting information section). 220 240 260 280 300 -3 -2 -1 0 1 2 3ctDNA ctDNA+bmim-AOT(2x10-3M) ctDNA+bmim-AOT(4x10-3M) ctDNA+bmim-AOT(6x10-3M) obs (mdeg) (nm) A Page 18 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 19 220 240 260 280 300 -3 -2 -1 0 1 2 3B obs (mdeg) (nm) ctDNA ctDNA+hmim-AOT(2x10-3M) ctDNA+hmim-AOT(4x10-3M) ctDNA+hmim-AOT(6x10-3M) Figure 4. CD spectra of ctDNA interacting with the vesicular system water/bmim-AOT (A) and water/hmim-AOT (B). [ctDNA] = 5x10-5 M. T = 25 °C. Figure 4 shows changes in the DNA CD spectra, which indicates that bmim- AOT and hmim-AOT vesicles interact with the polynucleotide. The intensity of both bands decreases upon increasing surfactant concentration, although the more substantial changes occur in the negative band. These results indicate that the vesicles interact with the DNA, partially modifying the polynucleotide conformation.56 It is interesting to note that no changes in the DNA CD spectrum are observed in the solutions of bmim-Cl and hmim-Cl with DNA (Figure S4), indicating that the cationic precursors do not interact with the DNA. Additionally, the DNA CD spectrum in presence of the Na-AOT surfactant were taken (results not shown), and the DNA was not affected. This would indicate that it is necessary for the counterion to have a hydrophobic region so that it can interact with the polynucleotide. In order to compare between both vesicular systems, ellipticity ( of the negative band, at  = 246 nm, was plotted in Figure 5 at different surfactant concentrations. Page 19 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 20 0.0 1.0x10-3 2.0x10-3 3.0x10-3 4.0x10-3 5.0x10-3 6.0x10-3 -3.0 -2.5 -2.0 -1.5 -1.0 ctDNA ctDNA+bmim-AOT ctDNA+hmim-AOT obs (mdeg) [Surfactant] (M) Figure 5. Variation of obs with [surfactant] in the vesicular system water/bmim-AOT and water/hmim-AOT. [ctDNA] = 5x10-5 M. T = 25 °C.  = 246 nm. This Figure shows that hmim-AOT vesicles interact more strongly with DNA than bmim-AOT vesicles. One would expect stronger electrostatic interactions between bmim-AOT and DNA than for the hmim-AOT and DNA based on the zeta potential values listed in Table 2. However, hydrophobic interactions, which play a key role in the interactions between DNA and other species32,57 will be stronger as the longer is the hydrophobic tail of the surfactant. Taking this into account, results in Figures 4 and 5 could be explained by the influence of the surfactant tail length on the vesicles:DNA interactions. In future work the interactions between the IL-like surfactants and DNA, and the cytotoxicity of these aggregates will be investigated in more depth since the design of biocompatible nanocarriers for genetic material is relevant in regard to gene transfection. Page 20 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 21 CONCLUSIONS The results obtained in this investigation show that the replacement of Na+ by bmim+ and hmim+ in the Na-AOT molecule drastically affects the physicochemical properties of the aggregates formed by the surfactant in water. The ionic liquids likesurfactants bmim-AOT and hmim-AOT have the ability to form spontaneously unilamellar vesicles in water. Hmim-AOT vesicles are smaller, with less negative surface charge, greater rigidity of the bilayer and greater capacity to solubilize molecules of low polarity, in comparison to bmim-AOT vesicles. It was also observed that hmim-AOT vesicles interact more with the DNA than bmim-AOT vesicles. This suggests the importance of hydrophobic interactions in the formation of surfactant-DNA complexes. The study of the interaction of surfactants with DNA deserves further investigation, since these aggregates could be efficient non-viral vectors in the transfection of genes. ASSOCIATED CONTENT Supplementary Information. NMR and FT-IR characterization of hmim-AOT, DLS data of aqueous solutions of bmim-AOT and hmim-AOT, circular dichroism spectra of ctADN whit bmim-Cl and hmim-Cl in water. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interests. Page 21 of 30 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 22 ACKNOWLEDGEMENTS We gratefully acknowledge the financial support for this work by the Consejería de Conocimiento, Investigación y Universidad de la Junta de Andalucía (P12-FQM- 1105), FQM-274, the Consejo Nacional de Investigaciones Científicas y Técnicas (PIP CONICET 112-2015-0100283), Universidad Nacional de Río Cuarto (PPI-UNRC 2016- 2018), Agencia Nacional de Promoción Científica y Técnica (PICT 2012-0232, PICT 2012-0526, PICT 2015-0585 and PICT-2015-2151), and Ministerio de Ciencia y Tecnología, Gobierno de la Provincia de Córdoba (PID 2013). N.M.C., J.J.S. and R.D.F. hold a research position at CONICET. C.M.O.L. thanks CONICET for a research fellowship and Asociación Universitaria Iberoamericana de Postgrado (AUIP) and Consejería de Economía y Conocimiento de la Junta de Andalucía for a fellowship of the Programa de Becas de Movilidad Académica de la AUIP, with which Dr. Lépori made the research stay at the University of Seville (Spain). REFERENCES (1) Hallett, J. P.; Welton, T. Room-Temperature Ionic Liquids: Solvents for Synthesis and Catalysis. 2. Chem. Rev. 2011, 111 (5), 3508–3576. (2) Wasserscheid, P.; Welton, T. Ionic Liquids in Synthesis: Second Edition; Wasserscheid, P., Welton, T., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2008; Vol. 1. (3) Falcone, R. D.; Correa, N. M.; Silber, J. J.; Levinger, N. E. Ionic Liquids in Soft Confinement. In Ionic Liquid-Based Surfactant Science; John Wiley & Sons, Inc: Hoboken, NJ, 2015; pp 283–301. (4) Welton, T. Room-Temperature Ionic Liquids. 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