Freeze-Drying of Aqueous Solutions of Deep Eutectic Solvents: A

Apr 23, 2009 - Miguel Hern´andez, Elche. 03202, Alicante, Spain. Received November 26, 2008. Revised Manuscript Received March 9, 2009. This work ...
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Freeze-Drying of Aqueous Solutions of Deep Eutectic Solvents: A Suitable Approach to Deep Eutectic Suspensions of Self-Assembled Structures Marıa C. Gutierrez,*,† Marıa L. Ferrer,† C. Reyes Mateo,‡ and Francisco del Monte*,† †

Instituto de Ciencia de Materiales de Madrid (ICMM), Consejo Superior de Investigaciones Cientıficas (CSIC), Cantoblanco, 28049 Madrid, Spain, and ‡Instituto de Biologıa Molecular y Celular, Universidad Miguel Hern andez, Elche. 03202, Alicante, Spain Received November 26, 2008. Revised Manuscript Received March 9, 2009 This work describes how the preparation of deep eutectic solvents (DES) in its pure state can be accomplished through a simple approach based on the freeze-drying of aqueous solutions of the individual counterparts of DES. DES in its pure state obtained via freeze-drying are studied by 1H NMR, which reveals the formation of halide ion-hydrogenbond-donor supramolecular complexes (characteristic of DES), and by cryo-etch-SEM, which provides insight about the capability of aqueous solutions of DES to be segregated in DES and ice upon freezing. The paper also explores the suitability of the freeze-drying approach to incorporate organic self-assemblies (in particular, liposomes of ca. 200 nm) in DES with full preservation of their self-assembled structure. This is not a trivial issue given that amphiphilic molecules tend to be readily dissolved (hence, disassembled) in DES. The strategy proposed in this work is based on the freezedrying of aqueous solutions containing the individual counterparts of DES and the preformed liposomes (also known as large unilamellar vesicles or LUV). The simplicity of the method should also make it suitable for the incorporation of different self-assembled structures (such other types of vesicles and micelles) in DES in its pure state.

Introduction The principle of creating an ionic fluid by complexing a halide salt was first demonstrated for mixtures of quaternary ammonium salts with a range of amides1 and later extended to a wide variety of other hydrogen bond donors (such as acids, amines, and alcohols, among others).2-5 The charge delocalization occurring through hydrogen bonding between the ion pair is responsible for the observed freezing-point depression in the mixture as compared to the individual counterparts. These ionic fluids (so-called deep eutectic solvents, DES) have exhibited similar features to ambient temperature ionic liquids;6 e.g., DES are nonreactive with water, biodegradable, and excellent solvents for a wide variety of solutes such as, among others, the different substrates and enzymes of catalytic and biocatalytic interest.7-12 Because of these attractive features, both IL and DES have been extensively studied as media for organic and inorganic synthetic reactions and separations. Within this context, the incorporation of core-shell structure formed by amphiphilic molecules and polymers in IL and DES would open *Corresponding authors: Fax (+34) 91 372 0623; e-mail mcgutierrez@ icmm.csic.es or [email protected]. (1) Abbott, A. P.; Capper, G.; Davies, D. L.; Rasheed, R. K.; Tambyrajah, V. Chem. Commun. 2003, 70–71. (2) Abbott, A. P.; Boothby, D.; Capper, G.; Davies, D. L.; Rasheed, R. K. J. Am. Chem. Soc. 2004, 126, 9142–9147. (3) Abbott, A. P.; Capper, G.; Davies, D. L.; Rasheed, R. K. Chem.;Eur. J. 2004, 10, 3769–3774. (4) Fukaya, Y.; Iizuka, Y.; Sekikawa, K.; Ohno, H. Green Chem. 2007, 9, 1155– 1157. (5) Parnham, E. R.; Drylie, E. A.; Wheatley, P. S.; Slawin, A. M. Z.; Morris, R. E. Angew. Chem., Int. Ed. 2006, 45, 4962–4966. (6) Abedin, S. Z. E.; Endres, F. Acc. Chem. Res. 2007, 40, 1106–1113. (7) Abbott, A. P.; Bell, T. J.; Handa, S.; Stoddart, B. Green Chem. 2005, 7, 705– 707. (8) Yang, Z.; Pan, W. Enzyme Microb. Technol. 2005, 37, 19–28. (9) Ueki, T.; Watanabe, M. Macromolecules 2008, 41, 3739–3749. (10) Zhao, H. J. Mol. Catal. B: Enzym. 2005, 37, 16–25. (11) Lee, S. H.; Dang, D. T.; Ha, S. H.; Chang, W.-J.; Koo, Y.-M. Biotechnol. Bioeng. 2008, 99, 1–8. (12) Gorke, J. T.; Srienc, F.; Kazlauskas, R. J. Chem. Commun. 2008, 1235– 1237.

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the possibility to provide well-defined hydrophobic and hydrophilic nanodomains in these solvents, thus leading to broader applications in aqueous chemistry. The interest in micellization of lipids, surfactants, and amphiphilic polymers in IL dates back to 1982, when the micellization of surfactants such as alkyltrimethylammonium bromides, alkylpyridinium bromides, and Triton X-100 and the phase transition of phospholipids such dipalmitoylphosphatidylethanolamine (DPPE), L-dipalmitoylphosphatidylcholine (DPPC), and β,γ-distearoylphosphotidylcholine (DSPC) were first studied in N-ethylammonium nitrate (EAN).13-16 In spite of the great interest in incorporating organic self-assemblies into IL, the number of works reporting on this topic is still limited; prior studies have employed EAN as IL17 or have focused on specific conditions (e.g., vesicles in ether-containing IL,18 liquid crystals containing functional mesogens specially designed for this specific purpose,19 or micelles of surfactants and pluronics in aqueous solutions of IL,20 among others21). It is worthy to note that the incorporation (and subsequent preservation) of self-assembled structures in this type of solvents has been demonstrated to be somehow difficult, most likely due to partial solubility of amphiphilic molecules which, ultimately, impedes their rearrangement into assembled structures. Actually, the (13) Evans, D. F.; Yamauchi, A.; Roman, R.; Casassa, E. Z. J. Colloid Interface Sci. 1982, 88, 89–96. (14) Evans, D. F.; Kaler, E. W.; Benton, W. J. J. Phys. Chem. 1983, 87, 533–535. (15) Tamura-Lis, W.; Lis, T. J.; Quinn, P. J. J. Phys. Chem. 1987, 91, 4625–4627. (16) Tamura-Lis, W.; Lis, T. J.; Quinn, P. J. Biophys. J. 1988, 53, 489–492. (17) Araos, M. U.; Warr, G. G. J. Phys. Chem. B 2005, 109, 14275–14277. (18) Nakashima, T.; Kimizuka, N. Chem. Lett. 2001, 31, 1018. (19) Fletcher, K. A.; Pandey, S. Langmuir 2004, 20, 33. (20) (a) Behera, K.; Pandey, S. Langmuir 2008, 24, 6462–6469. (b) Zheng, L.; Guo, C.; Wang, J.; Liang, X.; Chen, S.; Ma, J.; Yang, B.; Jiang, Y.; Liu, H. J. Phys. Chem. B 2007, 111, 1327–1333. (c) Zhang, S.; Li, N.; Zheng, L.; Li, X.; Gao, Y.; Yu, L. J. Phys. Chem. B 2008, 112, 10228–10233. (21) (a) Bai, Z.; Lodge, T. P. Langmuir 2008, 24, 5284–5290. (b) Ma, K.; Shahkhatuni, A. A.; Somashekhar, B. S.; Gowda, G. A. N.; Tong, Y. Y.; Khetrapal, C. L.; Weiss, R. G. Langmuir 2008, 24, 9843–9854. (c) Wang, L.; Chen, X.; Chai, Y.; Hao, J.; Sui, Z.; Zhuang, W.; Sun, Z. Chem. Commun. 2004, 2840–2841.(d) For a recent review, see also: Greaves, T. L.; Drummond, C. J. Chem. Soc. Rev. 2008, 37, 1709-1726.

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incorporation of self-assembled structures such as micelles and vesicles in DES (and different than those formed by DES themselves)22 remains largely unexplored. Under these circumstances, the design of a simple and standard chemical routine to incorporate self-assembled structures into DES (that is, suitable for different self-assembled structures in different DES or even IL), despite its tremendous interest, remains a challenge. Our work describes an easy procedure that allows the incorporation of self-assembled structures in DES in its pure state. Among different organic self-assemblies, we focused our study on the incorporation of liposomes. Liposomes are spherical closed vesicles of phospholipids bilayers with an entrapped aqueous phase and may consist of one or more bilayers (unilamellar or multilamellar, respectively) ranging in size from tens of nanometers to tens of micrometers. The number of suitable strategies for the formation and/or preservation of liposomes in IL and DES reported to date was quite limited (e.g., one for IL17 and none for DES). Note that rearrangement of lipids into vesicles by their direct dissolution in nonaqueous solvents such DES is certainly unlikely since entrapped water is required for liposome (also known as large unilamellar vesicles or LUV) formation.23 The most widely used method for LUV preparation is thin film hydration.24 Taking into consideration this requirement, a plausible strategy for incorporation of LUV in DES should be based on transitioning from aqueous chemistry to DES chemistry. Here, such transition is accomplished via freeze-drying of aqueous solutions containing preformed LUV besides the individual counterparts of DES. In particular, we have studied the preparation of urea-choline chloride and thiourea-choline chloride based DES (UCCl-DES and TUCCl-DES, respectively) in a 2:1 molar ratio. The success of this route resided in the achievement of two separate targets: (1) the formation of DES (that is, of halide ion-hydrogen-bond-donor supramolecular complexes) by freeze-drying of aqueous solutions of the individual counterparts (rather than by the typical preparation process of DES; e.g., thermal treatment in absence of water) and (2) the preservation of the self-assembled structure of LUV after the freeze-drying process. Thus, the formation of DES upon freeze-drying was first studied on aqueous solutions of urea (or thiourea) and choline chloride by 1H NMR spectroscopy. For this purpose, we first studied the stability of the halide ion-hydrogen-bond-donor supramolecular complexes in diluted DES. The capability of urea (or thiourea) and choline chloride to be segregated from ice upon freezing was studied by cryo-etch-SEM in aqueous solutions having different urea (or thiourea) and choline chloride concentrations. The suitability of this freeze-drying approach for the preparation of DES in its pure state containing monodisperse (ca. 200 nm diameter) LUV was finally demonstrated by cryoetch-SEM and confocal fluorescence microscopy.

Experimental part 1. Materials. Thiourea and choline chloride (from SigmaAldrich), and urea (from Fluka) were used as received. The phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was purchased from Avanti Polar Lipids, Inc. (22) (a) Cooper, E. R.; Andrews, C. D.; Wheatley, P. S.; Webb, P. B.; Wormald, P.; Morris, R. E. Nature (London) 2004, 430, 1012. (b) Greaves, T. L.; Weerawardena, A.; Krodkiewska, I.; Drummond, C. J. J. Phys. Chem. B 2008, 112, 896–905. (c) Drylie, E. A.; Wragg, D. S.; Parnham, E. R.; Wheatley, P. S.; Slawin, A. M. Z.; Warren, J. E.; Morris, R. E. Angew. Chem., Int. Ed. 2007, 46, 7839–7843. (d) Jing, B.; Chen, X.; Zhao, Y.; Wang, X.; Ma, F.; Yue, X. J. Mater. Chem. 2009, DOI: 10.1039/b818006g. (23) Lasic, D. D. Biochem. J. 1988, 29, 335–348. (24) Bangham, A. D.; Standish, M. M.; Watkins, J. C. J. Mol. Biol. 1965, 13, 238–252.

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(Alabaster, AL). The fluorescence probe, 5-buty1-4,4-difluoro4-bora-3a, 4a-diaza-s-indacene-3-nonanoic acid (C4-BODIPY 500/510 C9), was purchased from Molecular Probes. Water was distilled and deionized. 2. Thermal Procedure for Deep Eutectic Solvent Formation (DES): Preparation of UCCI-DES and TUCCI-DES. Both UCCI-DES and TUCCI-DES were formed by heating their components in a 2:1 molar ratio (urea (12.01 g, 0.2 mol) with choline chloride (13.96 g, 0.1 mol) and thiourea (15.22 g, 0.2 mol) with choline chloride (13.96 g, 0.1 mol), respectively) to 80 °C and stirring until a homogeneous liquid was formed. The resulting UCCI-DES and TUCCI-DES (in this case, temperature was kept above 69 °C to have TUCCI-DES in its liquid state)1 were placed in capillary tubes for 1H NMR characterization (see Table 1). D2O dilutions of UCCI-DES and TUCCIDES having 5, 10, 43 and 86 wt % solute content (so called UCCI-DESn, where n stands for the solute content of the original aqueous solution; (e.g.) 5, 10, 43, and 86 wt %, respectively) were also prepared for 1H NMR characterization (see Table 1). 3. Preparation of D2O Solution of U+CCI and TU+CCI. D2O solutions of U+CCI (or TU+CCI) in a 2:1 molar ratio and having 5, 10, and 43 wt % solute contents (so-called U+CCLn, where n stands for the solute content of the original aqueous solution; e.g. 5, 10, and 43 wt %, respectively ) were prepared by mixing separated D2O solutions of urea (or thiourea) and choline chloride (see table S1 Supporting Information for details). 4. Freeze-Drying Procedure for Deep Eutectic Solvent Formation (FD-DES) from Aqueous Solutions of Urea (or Thiourea) and Choline Chloride. Aqueous solutions of urea (or thiourea) and choline chloride in a 2:1 molar ratio and having 5 wt % solute contents were prepared by mixing separated aqueous solutions of urea (or thiourea) and choline chloride. For instance, 10 mL of an aqueous solution of urea 4.6 wt % was mixed with 10 mL of an aqueous solution of choline chloride (5.4 wt %) to obtain 20 mL of an aqueous solution of urea and choline chloride (5 wt %) The resulting aqueous solutions were frozen (at 77 and 253 K) and subsequently freeze-dried for the achievement of clear viscous liquids so called FD77-UCCI-DES and FD235-UCCI-DES (FD77TUCCI-DES and FD235-TUCCI-DES for thiourea samples) depending on the freezing temperature, respectively. The resulting viscous liquids were placed in capillary tubes for 1H NMR characterization (see Table 1). 5. Preparation of Non-Aqueous Deep Eutectic Suspensions of Liposomes. 5.1. Liposome Formation for CryoEtch-SEM Studies. DMPC phospholipids (33 mg) were dissolved in a round-bottom flask with a small amount of chloroform. The resulting clear solution was submitted to vacuum for 2 h for solvent evaporation and formation of a dry lipid film. Rehydration of the dried lipid films in buffer Tris 0.02 M pH 7 (0.8 mL) heated above the liposome phasetransition temperature (23 oC) resulted in the formation of multilamellar lipid vesicles (MLV).Liposomes (unilamellar lipid vesicles, LUV) of 200 nm mean diameter were obtained by a 20fold extrusion process of the MLV Suspension through a carbonate membrane filter in an Avanti Miniextruder suited at 40 oC. The concentration of the resulting DMPC solution was 60 mM. Afterwards, 0.04 mL of DMPC solution 60 mM was added to 0.01 mL of an aqueous solution of urea (or thiourea) and choline chloride having 20 wt % solute content so that the final solute content was 4 wt %. The resulting suspensions were studied by cryo-etch-SEM 24 h after their preparation. Langmuir 2009, 25(10), 5509–5515

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Figure 1. 1H NMR of (a) UCCl-DES, (b) FD77-UCCl-DES, (c) FD253-UCCl-DES, (d) TUCCl-DES, (e) FD77-TUCCl-DES, and (f) FD253-TUCCl-DES.

5.2. Liposome Formation for Confocal Fluorescence Microscopy Studies. The green fluorescent fatty acid Bodipy 500/510 was the fluorescence probe used for confocal fluorescence microscopy analysis. In this case, aliquots of Bodipy in methanol (5.9 μL, 1 mM) were directly added to the DMPC phospholipid (4.2 mg) dissolution in chloroform prior to the evaporation process. Bodipy 500/510 doped liposomes of 200 nm mean diameter were obtained after extrusion process, as described before. In this case, the resulting concentration of DMPC solution was 5.9 mM, and the final probe:lipid molar ratio was 1:1000. Subsequently, 1 mL of an aqueous solution of urea and choline chloride having 20 wt % solute content was added to 4 ml of a 5 μM DMPC liposome suspension in buffer Tris 0.01 M pH 7. The resulting aqueous suspension was freezedried and studied by confocal fluorescence microscopy for the observation of liposomes suspended in FD-UCCI-DES. 6. Samples Characterization. Thermogravimetric analyses (TG) were carried out in a SEIKO TG/ATD 320 U, SSC 5200 (Seiko Instruments) from room temperature to 350 oC at a heating rate of 10 oC/min and under nitrogen flow (100 mL/min). Langmuir 2009, 25(10), 5509–5515

For cryo-etch-SEM experiments, aqueous solutions were placed in the sample holder of a cryotransfer system (Oxford CT1500), plunged into subcooled liquid nitrogen, and then transferred to a preparation unit via an air lock transfer device. The frozen specimens were cryofractured and transferred directly via a second air lock to the microscope cold stage, where they were etched for 2 min at -90oC. After ice sublimation and in the preparation unit, the etched surfaces were sputter-coated with gold for 10 min at a sputter current of 10 mV. The thickness of the resulting gold film was within the 5–10 nm range, which allows for the undistorted observation of liposomes of ca. 200 nm diameter. Samples were subsequently transferred onto the cold stage of the scanning electron microscope chamber. Fractured surfaces were observed with a DSM 960 Zeiss scanning electron microscope at -135 oC under the following conditions: acceleration potential, 15 kV; working distance, 15 mm; and probe current, 5–10 nA. Confocal fluorescence microscopy was performed with a Radiance 2100 (Bio-Rad) laser scanning system on a Zeiss Axiovert 200 microscope. Micrographs were taken with 10 μm deep in focus and 488 nm excitation wavelength. Light emission was recorded for DOI: 10.1021/la900552b

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Table 1. 1H NMR Spectroscopy Data of Urea/Choline Chloride and Thiourea/Choline Chloride Based DES (UCCl-DES and TUCCl-DES, Respecively) and of the Individual Components of DES (U+CCl and TU+CCl)a δ (ppm) b

(1)b choline

(2) urea (thiourea) sample

HDO

R(-NH2)2

HO

CH2

CH2

N(CH3)3

c

CCl 4.22 (1.9H) 3.53 (2H) 2.99 (2H) 2.67 (9H) 5.56 (8.1H) 4.74 (0.7H) 3.40 (2H) 2.96 (2H) 2.64 (9H) UCCl-DES 3.90 (0.5H)d UCCl-DES86 4.03 (4.2H) 5.44 (5.5H) 3.41 (2H) 2.93 (2H) 2.62 (9H) UCCl-DES43 4.09 (8.2H) 5.26 (1.4H) 3.47 (2H) 2.94 (2H) 2.63 (9H) UCCl-DES10 4.20 (10.5H) 3.52 (2H) 2.99 (2H) 2.67 (9H) UCCl-DES5 4.20 (11.4H) 3.52 (2H) 2.99 (2H) 2.67 (9H) TUCCl-DES 6.66 (8.5H) 4.08 (0.8H) 3.43 (2H) 2.97 (2H) 2.65 (9H) TUCCl-DES86 3.65 (3.8H) 6.78 (5.0H) 3.44 (2H) 2.95 (2H) 2.63 (9H) TUCCl-DES43 3.98 (8.1H) 6.61 (1.0H) 3.46 (2H) 2.94 (2H) 2.62 (9H) TUCCl-DES10 4.18 (10.4H) 3.51 (2H) 2.97 (2H) 2.66 (9H) TUCCl-DES5 4.22 (11.4H) 3.53 (2H) 2.98 (2H) 2.67 (9H) U+CCl43 4.09 (7.6H) 5.28 (1.4H) 3.48 (2H) 2.95 (2H) 2.64 (9H) U+CCl10 4.21 (9.6H) 3.52 (2H) 2.99 (2H) 2.67 (9H) U+CCl5 4.22 (9.4H) 3.52 (2H) 2.99 (2H) 2.67 (9H) TU+CCl43 3.96 (7.4H) 6.59 (0.7H) 3.44 (2H) 2.92 (2H) 2.61 (9H) TU+CCl10 4.20 (8.4H) 3.53 (2H) 2.99 (2H) 2.68 (9H) TU+CCl5 4.22 (9.4H) 3.53 (2H) 2.99 (2H) 2.67 (9H) d 5.56 (8.0H) 4.81 (0.9H) 3.40 (2H) 2.96 (2H) 2.65 (9H) FD77-UCCl-DES 3.87 (0.9H) d 5.55 (8.2H) 4.81 (0.9H) 3.41 (2H) 2.96 (2H) 2.65 (9H) FD253-UCCl-DES 3.87 (0.9H) FD77-TUCCl-DES 6.68 (8.4H) 4.07 (0.7H) 3.42 (2H) 2.97 (2H) 2.65 (9H) FD253-TUCCl-DES 6.64 (8.2H) 4.08 (0.7H) 3.43 (2H) 2.95 (2H) 2.64 (9H) a Spectra of non-diluted UCC1-DES and TUCC1-DES samples were recorded using CHCl3 and DMSO, respectively, as external references,while dioxane was always used as external reference in D2O diluted samples. b The molar ratio of urea (or thiourea) to choline chloride was 2:1 in every sample. c The CCl solute content in D2O was 6.5 wt %. d Traces of water were detected in UCCl-DES and FD-UCCl-DES given its hygroscopic character. This was not the case for TUCCl-DES and FD-TUCCl-DES because the spectra were carried out at 100 °C.

Figure 2. Details of downfield shifts of HDO (grease box) and HO-CH2-CH2-N(CH3)3 (blue box) signals as dilution increases from 86, to 43, to 10 up to 5 wt % (e.g., UCCl-DES86, UCCl-DES43, UCCl-DES10, and UCCl-DES5 in left column and TUCCl-DES86, TUCClDES43, TUCCl-DES10, and TUCCl-DES5 in right column).

wavelengths ranging from 505 to 575 nm. 1H NMR spectra (500 MHz) were recorded using a Bruker spectrometer DRX-500. Nondiluted UCCI-DES and TUCCI-DES samples were placed in capillary tubes using chloroform (CHCl3) and dimethyl sulfoxide (DMSO) as external reference, respectively. The 1H NMR spectrum of nondiluted TUCCI-DES was carried out at 100 oC, well above its melting point (69 oC)1. In diluted samples, D2O was used as solvent (the deuterium signal was used for locking and shimming the sample) and dioxane as the external reference. 5512

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Results and Discussion As mentioned in the Introduction, liposomes are spherical closed vesicles of phospholipids bilayers with an entrapped aqueous phase and may consist of one or more bilayers (unilamellar or multilamellar, respectively) ranging in size from tens of nanometers to tens of micrometers. Liposomes as models of living cells present an enormous opportunity for incorporating cellular properties into these artificial vesicles, hence the Langmuir 2009, 25(10), 5509–5515

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Figure 3. Cryo-etch-SEM micrographs of (from left to right, and from top to bottom) UCCl-DES5, TUCCl-DES5, UCCl-DES10, TUCClDES10, UCCl-DES43, and TUCCl-DES43. Bars are 20 μm. Inset shows a detail of the TUCCl-DES5 structure (bar is 5 μm).

idea of developing liposomes for biosensors and bioelectronic devices.25 For instance, the presence of liposomes in DES would also open the path for the incorporation of transmembrane proteins.26 Transmembrane proteins act as light absorptiondriven transporters, oxidoreduction-driven transporters, electrochemical potential-driven transporters, and ion channels27 and immersed in the membrane-like structure of liposomes could offer interesting properties in combination with DES. (25) (a) Rangin, M.; Basu, A. J. Am. Chem. Soc. 2004, 126, 5038–5039. (b) Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L.; Kool, E. T.; Boxer, S. G. J. Am. Chem. Soc. 2005, 127, 1356–1357. (c) Santos, M.; Roy, B. C.; Goicoechea, H.; Campiglia, A. D.; Mallik, S. J. Am. Chem. Soc. 2004, 126, 10738–10745. (26) (a) Lee, A. G. Biochim. Biophys. Acta 2003, 1612, 1–40. (b) Rigaud, J. L.; Bluzat, A.; Buschlen, S. Biochem. Biophys. Res. Commun. 1983, 111, 373–382. (27) (a) Mombaerts, P. Science 1999, 286, 707–711. (b) Bowie, J. U. Curr. Opin. Struct. Biol. 2001, 11, 397–402.

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In this work, we have studied LUV ranging from 100 to 400 nm in diameter which are obtained by extrusion of multilamellar vesicles (MLV) through small pore filters. The classical method for preparation of MLV is thin film hydration, which is a threestep process consisting of (1) the lipids dissolution in an organic solvent, (2) the evaporation of the solvent to form a thin film at the bottom of the flask, and (3) the hydration of the thin film giving rise to the spontaneous formation of MLV ranging in size from several tenths of a micrometer to tens of micrometers. Taking into consideration that the presence of water is required for liposome formation, one plausible route for LUV incorporation in DES would be through their direct mixing in aqueous solutions. Unfortunately, DES tends to become a simple solution of its individual counterparts upon dilution, which is an undesired event given that some of the above-mentioned features offered by DES in its pure state (i.e., in the absence of water) might be DOI: 10.1021/la900552b

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(at least partially) missed. The comparison between the 1H NMR spectra of DES in its pure state and those of aqueous solutions of DES having different concentrations (e.g., 86, 43, 10, and 5 wt %) confirmed this issue. The spectra of nondiluted samples (e.g., UCCl-DES and TUCCl-DES) showed the characteristic signals of choline chloride protons and urea (or thiourea) (see Figure 1a,d and Table 1). One of the most significant differences in the spectra of UCCl-DES86 and TUCCl-DES86 was the appearance of the HDO signal (Figure 2 and Table 1), which is indicative of partial urea and thiourea protons exchange with D2O. This exchange was also reflected in the intensity of the urea and thiourea signals, i.e., below the stoichiometrically expected, that is, 5H rather than 8H (Figure 2 and Table 1). Interestingly, the chemical shift of the HDO signal (up to 0.2 ppm upfield shifted as compared to single choline chloride, CCl in Figure S1 and Table 1) revealed a somehow weak hydrogen-bonding structure in HDO, a consequence of the still significant presence of halide ion-hydrogen-bond-donor supramolecular complexes in both UCCl-DES and TUCCl-DES (that is, urea-choline chloride and thiourea-choline chloride ion pairs, respectively) at those dilutions (e.g. 86 wt %).28 Further dilution promoted the rupture of halide ion-hydrogen-bond-donor supramolecular complexes. Thus, the 1H NMR spectra of D2O solutions of UCCl-DES and TUCCl-DES having 43, 10, and 5 wt % solute content (e.g., UCCl-DES43, UCClDES10, UCCl-DES5, TUCCl-DES43, TUCCl-DES10, and TUCCl-DES5) exhibited a downfield shift of the choline chloride protons (up to 0.1 ppm; see Figure 2 and Table 1), a feature indicative of the increased hydration of choline chloride molecules. The rupture of the urea-choline chloride and thioureacholine chloride ion pairs in diluted DES was corroborated by the increase of the number of urea and thiourea protons interchanged with D2O; larger as DES concentration decreases. The chemical downfield shift of the HDO signal (i.e., up to 0.2 ppm for UCClDES5 and 0.6 ppm for TUCCl-DES5) was also indicative of such rupture. It is worth noting that downfield shifts in HDO signals are consequence of the formation of hydrogen bond structures in water, a feature that is favored by the presence of free urea and thiourea molecules (hence, noninteracting with choline chloride molecules) in solution.29 On the basis of the above results, dilution of DES (equal to or below 43 wt %) was not a valid approach if one desires to preserve the halide ion-hydrogen-bond-donor supramolecular complexes characteristic of DES. Ultimately, dilution of DES was equivalent to simple dilution of their individual counterparts as demonstrated the 1H NMR spectra (Figures S2 and S3) of D2O solutions of urea and choline chloride prepared separately and subsequent mixed in a 2:1 molar ratio and having 43, 10, and 5 wt % solute content (so-called U+CCln or TU+CCln, where n stands for the solute content, in Table 1). If one could recover the urea-choline and thiourea-choline ion pairs upon water elimination, the incorporation of LUV in DES in its pure state via mixing of aqueous solutions containing LUV and DES counterparts would be plausible. Among different alternatives explored for water elimination (e.g., rotaevaporation), freeze-drying was the selected method for dehydration given its capability (with the help of cryoprotectants) for the preservation of membrane-like structures. An interesting tool for the study of freeze-drying processes is (28) Gutowski, K. E.; Broker, G. A.; Willauer, H. D.; Huddleston, J. G.; Swatloski, R. P.; Holbrey, J. D.; Rogers, R. D. J. Am. Chem. Soc. 2003, 125, 6633. (29) (a) Ma, J.-H.; Guo, C.; Tang, Y.-L.; Chen, L.; Bahadur, P.; Liu, H.-Z. J. Phys. Chem. B 2007, 111, 5155. (b) Hebling, C. M.; Thompson, L. E.; Eckenroad, K. W.; Manley, G. A.; Fry, R. A.; Mueller, K. T.; Strein, T. G.; Rovnyak, D. Langmuir 2008, 24, 13866–13874.

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Figure 4. TG analysis of (a) UCCl-DES (dashed line) and FDUCCl-DES (solid line) and (b) TUCCl-DES (dashed line) and FDTUCCl-DES (solid line).

cryo-etch-SEM.30 In cryo-etch-SEM experiments, the aqueous solution was first plunge frozen by immersion in subcooled liquid nitrogen, that is, liquid nitrogen at vacuum pressure. The sample temperature was subsequently raised to -90 °C, which allowed exposed ice to sublimate (etching). This temperature (ca. 47 °C above the Tg of water) favored the formation of crystalline ice, which readily frees itself of any dissolved solute. Cryo-etch-SEM micrographs of aqueous solutions of UCCl-DES and TUCClDES with solute contents ranging from 5 to 43 wt % (Figure 3) revealed the formation of “fence”-like structures that consisted of solutes (in this case, UCCl-DES and TUCCl-DES) concentrated at the surrounding empty areas where ice originally resided. This was also the case for aqueous solutions of U+CCl and TU+CCl (Figure S4). Interestingly, analogue structures have been observed for aqueous solutions of ionic salts like NaCl31 which, as well as IL and DES,32 are characterized by some (depending on concentration) ice avoiding capability.33 Actually, similarities between IL and ionic salts are not rare; for instance, the importance of dispersion and induction interactions in IL has recently been compared to those of NaCl.34 The efficiency of the freeze-drying process to obtain DES in its pure state was further corroborated by the similarities (30) (a) Menger, F. M.; Seredyuk, V. A.; Apkarian, R. P.; Wright, E. R. J. Am. Chem. Soc. 2002, 124, 12408–12409. (b) Ferrer, M. L.; Esquembre, R.; Ortega, I.; Mateo, C. R.; del Monte, F. Chem. Mater. 2006, 18, 554–559. (c) Gutierrez, M. C.; Garcia-Carvajal, Z. Y.; Jobbagy, M.; Rubio, F.; Yuste, L.; Rojo, F.; Ferrer, M. L.; del Monte, F. Adv. Funct. Mater. 2007, 17 3505–3513. (31) Menger, F. M.; Galloway, A. L.; Chlebowski, M. E.; Apkarian, R. P. J. Am. Chem. Soc. 2004, 126, 5987–5989. (32) Byrne, N.; Wang, L.-M.; Belieres, J.-P.; Angell, C. A. Chem. Commun. 2007, 2714–2716. (33) Malmgren, F. Sci. Results 1927, 1. (34) Zahn, S.; Uhlig, F.; Thar, J.; Spickermann, C.; Kirchner, B. Angew. Chem., Int. Ed. 2008, 47, 3639–3641.

Langmuir 2009, 25(10), 5509–5515

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Article

Figure 5. Cryo-etch-SEM micrographs of LUV (ca. 200 nm diameter) suspended in UCCl-DES4 (left, bar is 20 μm) and TUCCl-DES4 (right, bar is 2 μm).

membrane-like structure characteristic of liposomes; i.e., the use of the hydrophobic fluorescent probe Bodipy 500/510 (hence, specifically allocated at the hydrophobic side of the membrane) allowed the observation of O-ring like structures, that is, spherical vesicles (Figure 6). It is worth noting that the polydispersion observed at confocal fluorescence microscope was wider than that at cryo-etch-SEM given the difficulties to impede some membrane fusion among adjacent liposomes in concentrated suspensions. Taking into account that the preservation of the membrane-like structures upon freeze-drying requires of cryoprotection,30b,35,36 the above-described results also revealed the cryoprotecting character of DES.

Conclusions

Figure 6. Confocal fluorescence micrograph of LUV (ranging from 200 to 500 nm diameter) suspended in FD-UCCl-DES (bar is 2 μm).

among the TG analysis of the original DES (i.e., prepared by thermal treatment) and the mixture resulting after freeze-drying an aqueous solution of DES (FD-DES, see Figure 4). Finally, 1H NMR spectroscopy corroborated freeze-drying of any aqueous solution having the individual counterparts of DES was indeed a valid route for the achievement of DES in its pure state; i.e., both the chemical shifts and the H number assigned to every signal of the 1H NMR spectra of FD-DES samples (see Figure 1 and Table 1) were identical to that of original DES. The capability to obtain DES in its pure state by freeze-drying of aqueous solutions of DES and of the individual counterparts of DES opened the possibility for the incorporation of LUV. As mentioned above, one could easily incorporate LUV in an aqueous solution (which, eventually, would also contain urea and choline chloride) and subsequently submit such aqueous solution to freeze-drying for water sublimation. The preservation of the characteristic self-assembled arrangement of LUV (e.g., spherical unilamellar vesicles) during and after freeze-drying was studied by cryo-etch-SEM and confocal fluorescence microscopy, respectively. At the frozen state, cryo-etch-SEM allowed the observation of “fence”-like structures, for both FD-UCClDES and FD-TUCCl-DES, made of spherical particles of ca. 200 nm (Figure 5). Confocal fluorescence microscopy not only corroborated the presence of liposomes ranging from 200 to 500 nm diameter, but also confirmed the preservation of the Langmuir 2009, 25(10), 5509–5515

In summary, we have demonstrated that the above-described freeze-drying process offers not only the possibility of incorporation of organic self-assemblies like LUV in DES in its pure state (with potential utility in catalytic applications) but also an alternative and easy procedure for DES preparation from aqueous solutions of their individual counterparts. We consider that the combined use of DES, offering special properties to the reaction medium (mostly in terms of viscosity, fluidity, polarity, ionic strength, etc.), and micelles and vesicles, acting as nanoreactors and capsules, could be of great interest in a number of emerging applications, such as catalytic, biocatalytic, or pharmaceutical, among others. Moreover, the incorporation of LUV in DES also opens the path for the unexplored study of transmembrane proteins in DES. Transmembrane proteins act as light absorption-driven transporters, oxidoreduction-driven transporters, electrochemical potential-driven transporters, and ion channels and immersed in the membrane-like structure of LUV could offer interesting properties in combination with DES. Acknowledgment. This work was supported by MAT 2006-02394, MAT2008-05670, 200660F011, and 200760I009 Projects. We also acknowledge TPA Inc. for financial support. M.C.G. acknowledges MEC for a R&C research contract. F. Pinto is acknowledged for valuable support with cryoetch-SEM experiments. Supporting Information Available: Further experimental details, 1H NMR spectra, and cryo-etch-SEM micrographs. This material is available free of charge via the Internet at http://pubs.acs.org. (35) Sun, W. Q.; Leopold, A. C.; Crowe, L. M.; Crowe, J. H. Biophys. J. 1996, 70, 1769–1776. (36) Ferrer, M. L.; Garcia-Carvajal, Z. Y.; Yuste, L.; Rojo, F.; del Monte, F. Chem. Mater. 2006, 18, 1458–1463.

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