pubs.acs.org/Langmuir © 2010 American Chemical Society
Synthesis and Physicochemical Characterization of New Twin-Tailed N-Oxide Based Gemini Surfactants )
Federico Bordi,† Giorgio Cerichelli,*,‡ Nadia de Berardinis,‡ Marco Diociaiuti,§ Luisa Giansanti,^ Giovanna Mancini,*,^, and Simona Sennato#
)
† Dipartimento di Fisica and INFM-CRS SOFT, Universit a degli Studi di Roma “La Sapienza”, Piazzale A. Moro 5, 00185 Roma, Italy, ‡Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universit a degli Studi de L’Aquila, UdR INCA, Via Vetoio, 67010 Coppito Due, L’Aquila, Italy, §Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanit a, V. le Regina Elena 299, 00161 Roma, Italy, ^CNR, Istituto di Metodologie Chimiche;Sezione Meccanismi di Reazione and Dipartimento di Chimica, Universit a degli Studi di Roma “La Sapienza”, P. le Aldo Moro 5, 00185 Roma, Italy, Centro di Eccellenza Materiali Innovativi Nanostrutturali per Applicazioni Cliniche, Fisiche e Biomediche, and #Dipartimento di Fisica and CNISM, Universit a degli Studi di Roma “La Sapienza”, Piazzale A. Moro 5, 00185 Roma, Italy
Received July 17, 2009. Revised Manuscript Received March 15, 2010 New gemini surfactants (GSs) constituted by two double alkyl chain (from 7 to 17 methylenic units) N-oxide monovalent surfactants joined by a PEG spacer of different length (from 3 to 21 ethylene glycol units), thus combining the properties of both N-oxide and GS surfactants, were synthetized and characterized. The different hydrophilic/ hydrophobic balance of the molecular structure strongly influences the morphology and the electrical features of the aggregates. Despite the zwitterionic nature of the polar head groups, all the aggregates are characterized by positive potential thus suggesting protonation at the interface; however, the extent of protonation was shown to strongly depend on the length of the alkyl chain and of the spacer.
Introduction Gemini surfactants1,2 (GSs) are amphiphilic molecules that contain two head groups and two aliphatic chains, linked by a rigid3-6 or flexible7,8 spacer. Their molecular structure confers them very peculiar physicochemical properties compared to the corresponding monovalent surfactants. In fact, they typically show highly superior surfactant properties with respect to the corresponding conventional amphiphiles; for example, surface activity can be increased 1000-fold. Moreover, GSs are characterized by lower critical micellar concentration (cmc) values, higher solubilization power, and hydrotropy with respect to the corresponding monovalent surfactants. The higher surface activity of GSs is advantageous for their applications in the industry for detergency and emulsification and involves the use of smaller amounts of raw material for synthesis and the handling of less manufacturing and byproduct, thus ending in a minor environmental impact.9 All these advantages make them of special interest also for biomedical applications, where they have been investigated as drug delivery *Corresponding authors. (G.M.) Telephone: 00390649913078. Fax: 003906490421. E-mail:
[email protected]. (G.C.) Telephone: 0039 0862433784. Fax: 0039 0862433753. E-mail:
[email protected]. At the time of publication, G.C.’s telephones were not working, as the Chemistry building cannot be used due to an earthquake; reconstruction is in course. (1) Menger, F. M.; Keiper, J. S. Angew. Chem. 2000, 112, 1980–1996. (2) Menger, F. M. Angew. Chem., Int. Ed. 2000, 39, 1906. (3) Menger, F. M.; Littau, C. A. J. Am. Chem. Soc. 1993, 115, 10083. (4) Zana, R.; Bennraou, M.; Rueff, R. Langmuir 1991, 7, 1072–1075. (5) Zana, R. Adv. Colloid Interface Sci. 2002, 97, 205–253. (6) Zana, R. J. Colloid Interface Sci. 2002, 248, 203. (7) Zana, R.; Talmon, Y. Nature 1993, 362, 228–230. (8) Karaborni, S.; Esselink, K.; Hilbers, P. A. J.; Smit, B.; Karthauser, J.; van Os, N. M.; Zana, R. Science 1994, 266, 254. (9) Hait, S. K.; Moulik, S. P. Curr. Sci. 2002, 82, 1100. (10) Bombelli, C.; Giansanti, L.; Luciani, P.; Mancini, G. Curr. Med Chem. 2009, 16, 171–183. (11) Wasungu, L.; Scarzello, M.; van Dam, G.; Molema, G.; Wagenaar, A.; Engberts, J. B. F. N.; Hoekstra, D. J. Mol. Med. 2006, 84, 774.
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systems10 and DNA carriers in transfection studies.11 Because of these features, though the family of GSs is relatively young (actually, they were first reported ∼40 years ago,12 but a large interest for these surfactants has spread ∼20 years later), there already are a large number of species for a whole range of applications. So far over 10 000 international patents on GSs have been filled, and investigations on many different applications are currently being reported. The manipulation of the basic structure of gemini can give an almost unlimited number of potential molecules, thus allowing extensive structure-activity studies aimed at identifying structural features necessary for the successful exploitation of GSs. Here we report the preparation of the new GSs (Scheme 1), composed of two (from 7 to 17 methylenic units) N-oxide monovalent surfactants bearing two alkyl chains and joined by a PEG spacer of different length (from 3 to 21 ethylene glycol units), and the physicochemical characterization of the aggregates they form in water. N-Oxide surfactants are in general biodegradable, show a low-to-moderate toxicity, and show good antioxidant13 and antimicrobial activity14 (both depending on the alkyl chain length). N-oxide surfactants are used in many cleaning formulations, in liquid bleach products, as antistatic agent in textile industry, as foam stabilizer in the rubber industry, as polymerization catalysts in polymer industry, in anticorrosion compositions, as lime soap dispersants, and as antibacterial agents in deodorant bars due to their compatible synergistic effect and environment friendly nature.15 The absence of counterions in (12) Bunton, C. A.; Robinson, L.; Schaak, J.; Stam, M. F. J. Org. Chem. 1971, 36, 2346. (13) Krasowska, A.; Piasecki, A.; Murzyn, A.; Sygler, K. Folia Microbiol. 2007, 52(1), 45. (14) Bukowsky, M.; Mlinarcik, D.; Ondrackova, V. Int. J. Immunopharm. 1996, 18(6/7), 423. (15) Singh, S. K.; Bajpay, M.; Tyagi, V. K. J. Oleo Sci. 2006, 55(3), 99.
Published on Web 03/31/2010
DOI: 10.1021/la1005067
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Bordi et al.
Scheme 1. Synthetic Pattern for the Preparation of GSs 1a-1ea
yield as yellow oil starting from 2b (1.0 g) following the same procedure described for 1a. 1 H NMR (CDCl3): δ 3.914 (4H, m, (C10)2NþO-CH2-); 3.539 (12H, m, -CH2OCH2-); 3.353 (8H, m, -CH2NþO-TEG); 1.654 (8H, bs, -CH2CH2NþO-TEG); 1.193 (56H, m, -(CH2)7-); 0.808 (12H, t, -CH3). 13 C NMR (CDCl3): δ 69.38; 64.41; 63.68; 30.88; 28.48; 28.27; 25.99; 21.98; 21.66; 13.07. Anal. Calcd for C48H100N2O5: C, 73.41; H, 12.83; N, 3.57; O, 10.19. Found: C, 73.26; H, 13.16; N, 3.33.
PEG 400 N,N-Di-n-decylamine-N-oxide (C10)2NþO-PEG400-NþO-(C10)2 (1c). The crude product 1c was ob-
a Key: (i) PBr3, pyridine; (ii) 2a, di-n-octylamine, 2b, 2c di-n-decylamine, 2d, 2e di-n-octadecylamine, Na2CO3, 120 C; (iii) H2O2, absolute ethanol, reflux.
N-oxide surfactants make them particularly interesting also in the field of cultural heritage (cleaning and protection of stonewall), in fact, the presence of salts can modify the capability of water to seep into the stone, causing serious damage to the monuments as a consequence of freeze and thaw cycles.16 Furthermore, they are investigated in pH controlled DNA condensation.17 Combining the properties of N-oxide surfactants with a GS type structure could further develop the potential of this compounds, extending the areas of their application or improving their performances.18
Experimental Section Materials. Phosphorus tribromide (PBr3), pyridine, tetraethylene glycol (TEG), poly(ethylene) glycol 400 (PEG 400), poly(ethylene) glycol 600 (PEG 600), poly(ethylene) glycol 1000 (PEG 1000), di-n-octylamine, di-n-decylamine, di-n-octadecylamine, and manganese oxide (MnO2) were purchased from SigmaAldrich and used without further purification. Solvents: HCl, Na2SO4, Na2CO3, and H2O2 40% m/v were purchased from Carlo Erba Reagenti. Bis(di-n-octylamine)bis(ethoxyethyl) Ether-N-oxide (C10)2NþO--TEG-NþO-(C10)2 (1a). H2O2, 0.33 mL (40% m/v, 3.9 mmol), was added dropwise with continuous stirring to a solution of 2a (1.0 g, 1.6 mmol) in absolute ethanol (0.33 mL) heated to reflux. After 4 h, the reaction mixture was cooled to room temperature and the excess of H2O2 decomposed with MnO2. The solid MnO2 was removed by filtration, washed with ethanol and the solvent was removed under reduced pressure. The residue was dissolved in diethyl ether and the solvent was removed under reduced pressure three times giving 1.1 g (90%) of crude product 1a as brownish oil. The product was purified by chromatography on silica gel eluting with chloroform-methanol (95:5, v/v) to give 750 mg of 1a as yellow oil (71% yield). 1 H NMR (CDCl3): δ 3.847 (4H, m, (C10)2Nþ(O-)CH2-); 3.451 (12H, m, -CH2OCH2-); 3.357 (8H, m, -CH2NþO-TEG-); 1.573 (8H, bs, -CH2CH2NþO-TEG-); 1.065 (40H, m, -(CH2)5-); 0.740 (12H, t, -CH3). 13 C NMR (CDCl3): δ 70.16; 70.01; 65.89; 64.89; 64.50; 31.41; 28.96; 28.81; 26.38; 22.87; 22.29; 13.74. Anal. Calcd for C40H84N2O5: C, 71.37; H, 12.58; N, 4.16; O, 11.88. Found: C, 71.17; H, 12.69; N, 3.98.
Bis(di-n-decylamine)bis(ethoxyethyl) Ether-N-oxide (C10)2NþO--TEG-NþO-(C10)2 (1b). 1b was obtained in 20% (16) Accardo, G.; Vigliano, G. Strumenti e materiali del restauro; Kappa: Rome, Italy, 1989. (17) Melnikova, Y. S.; Lindman, B. Langmuir 2000, 16, 5871. (18) Goracci, L.; Germani, R.; Rathman, J. F.; Savelli, G. Langmuir 2007, 23, 10525.
6178 DOI: 10.1021/la1005067
tained starting from 1.0 g of 2c following the same procedure described for 1a. The crude product 1c was dissolved in CH2Cl2 (5 mL), added dropwise with diethyl ether (200 mL) and kept in freezer for 24 h. The precipitate was collected by filtration, washed with diethyl ether, and dried under vacuum to give 300 mg of 1c as a yellow oil (30% yield). 1 H NMR (CDCl3): 3.861 (4H, m, (C10)2NþO-CH2-); 3.511 (30H, m, -CH2OCH2-); 3.267 (8H, m, -CH2NþO-TEG-); 1.608 (8H, bs, -CH2CH2NþO-TEG-); 1.146 (56H, m, -(CH2)7-); 0.763 (12H, t, -CH3). 13 C NMR (CDCl3): 70.29; 70.16; 65.47; 64.62; 31.61; 29.21; 29.02; 26.10; 22.77; 22.39; 13.82.
PEG 600 N,N-Di-n-octadecylamine-N-oxide (C18)2 NþO--PEG600-NþO- (C18)2 (1d). The crude compound 1d was obtained starting from 1.0 g of 2d following the same procedure described for 1a. Using the same method of purification described for 1c, 230 mg of 1d as pale yellow crystals were obtained (23% yield). 1 H NMR (CDCl3): δ 3.927 (4H, m, (C10)2NþO-CH2-); 3.564 (48H, m, -CH2OCH2-); 3.212 (8H, m, -CH2NþO-TEG-); 1.663 (8H, bs, -CH2CH2NþO-TEG-); 1.197 (120H, m, -(CH2)15-); 0.785 (12H, t, -CH3). 13 C NMR (CDCl3): δ 70.49; 70.31; 66.02; 64.74; 31.81; 29.58; 29.43; 26.58; 23.05; 22.56; 13.96.
PEG 1000 N,N-Di-n-octadecylamine-N-oxide (C18)2NþO--PEG1000-NþO-(C18)2 (1e). The crude compound 1e was obtained starting from 1.0 g of 2e following the same procedure described for 1a. Using the same method of purification as described for 1c, 230 mg of 1e as light yellow crystals were obtained (22% yield). 1 H NMR (CDCl3): δ 3.746 (4H, m, (C10)2NþO-CH2-); 3.578 (82H, m, -CH2OCH2-); 3.416 (8H, m, -CH2NþO-TEG); 1.362 (8H, bs, -CH2CH2NþO-TEG); 1.187 (120H, m, -(CH2)15-); 0.809 (12H, t, -CH3). 13 C NMR (CDCl3): δ 70.42; 64.63; 61.46; 31.73; 29.52; 29.35; 26.77; 22.14; 13.89. Bis(di-n-octylamine)bis(ethoxyethyl) Ether (2a). A solvent free mixture of di-n-octylamine (2.3 g, 9.4 mmol), 3a (1.5 g, 4.7 mmol), and Na2CO3 (1.0 g, 9.4 mmol) was stirred for 96 h at 120 C. The mixture was cooled to room temperature, added with 10 mL of distilled water and extracted with diethyl ether (3 30 mL). The organic layers were dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure gave compound 2a (2.4 g, 78% yield). 1 H NMR (CDCl3): δ 3.526 (12H, m, -CH2OCH2-); 2.569 (4H, t, (C10)2NþO-CH2-); 2.358 (8H, t, -CH2NþO-TEG-); 1.340 (8H, bs, -CH2CH2NþO-TEG-); 1.189 (40H, m, -(CH2)5-); 0.802 (12H, t, -CH3). 13 C NMR (CDCl3): δ 70.61; 70.40; 69.90; 54.91; 53.44; 31.79; 29.51; 29.38; 27.48; 27.28; 27.3; 22.57; 13.97. Bis(di-n-decylamine) bis(ethoxyethyl) Ether (2b). Compound 2b was obtained in 80% yield starting from 3a and di-ndecylamine following the same procedure described for 2a. 1 H NMR (CDCl3): δ 3.711 (12H, m, -CH2OCH2-); 3.172 (4H, t, (C10)2NþO-CH2-); 2.637 (8H, m, -CH2NþO-TEG-); 1.621 (8H, bs, -CH2CH2NþO-TEG-); 1.207 (56H, m, -(CH2)7-); 0.827 (12H, t, -CH3). Langmuir 2010, 26(9), 6177–6183
Bordi et al. C NMR (CDCl3): δ 70.31; 70.15; 60.42; 59.00; 58.14; 31.74; 29.49; 29.31; 29.16; 28.39; 27.16; 26.22; 22.56; 21.62; 14.01. 13
PEG 400 N,N-Di-n-decylamine (C10)2N-PEG400N(C10)2 (2c). Compound 2c was obtained in 94% yield starting from 3c and di-n-decylamine following the same procedure described for 2a. 1 H NMR (CDCl3): δ 3.566 (30H, m, -CH2OCH2-); 2.556 (4H, t, (C10)2NþO-CH2-); 2.347 (8H, t, -CH2NþO-TEG-); 1.291 (8H, bs, -CH2CH2NþO-TEG-); 1.177 (56H, m, -(CH2)7-); 0.795 (12H, t, -CH3). 13 C NMR (CDCl3): δ 70.46; 69.80; 54.82; 53.36; 31.75; 29.46; 29.17; 27.40; 27.24; 27.02; 22.51; 13.92.
PEG 600 N,N-Di-n-octadecylamine (C18)2N-PEG600N(C18)2 (2d). Compound 2d was obtained in 80% yield starting from 3d and di-n-octadecylamine following the same procedure described for 2a. 1 H NMR (CDCl3): δ 3.605 (48H, m, -CH2OCH2-); 2.607 (4H, t, (C10)2NþO-CH2-); 2.396 (8H, t, -CH2NþO-TEG); 1.423 (8H, bs, -CH2CH2NþO-TEG); 1.215 (120H, m, -(CH2)15-); 0.837 (12H, t, -CH3). 13 C NMR (CDCl3): δ 70.56; 70.40; 54.91; 53.44; 31.86; 29.63; 29.29; 27.50; 27.10; 22.61; 14.01.
PEG 1000 N,N-Di-n-octadecylamine (C18)2N-PEG1000N(C18)2 (2e). Compound 2e was obtained in 75% yield starting from 3d and di-n-octadecylamine following the same procedure described for 2a. 1 H NMR (CDCl3): δ 3.576 (82H, m, -CH2OCH2-); 3.426 (4H, t, (C10)2NþO-CH2-); 2.813 (8H, m, -CH2NþO-TEG); 1.637 (8H, bs, -CH2CH2NþO-TEG); 1.186 (120H, m, -(CH2)15-); 0.807 (12H, t, -CH3). 13 C NMR (CDCl3): δ 70.43; 64.64; 60.49; 59.20; 31.75; 29.53; 29.18; 26.23; 22.50; 13.91. Bis(5-bromoethoxyethyl) Ether (3a). First, 2.12 mL (0.02 mol) of PBr3 was cooled under nitrogen in a round-bottom flask to 0 C using an ice bath. A mixture of TEG (4a) (5.6 g, 0.03 mol) and pyridine (0.81 mL, 0.01 mol) was added to the solution slowly under stirring. The reaction mixture was allowed to warm to room temperature and to react for 8 h. The solution was added with water (50 mL), neutralized with HCl and extracted with CCl4 (3 50 mL) and the organic layer was dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure gave compound 4.1 g of 2a (42% yield). 1 H NMR (CDCl3): δ 3.749 (4H, t, BrCH2CH2-); 3.610 (8H, m, -OCHH2-); 3.413 (4H, t, BrCH2-). 13 C NMR (CDCl3): δ 70.96; 70.42; 70.30; 30.27. PEG 400 Dibromide (3c). Compound 3c was obtained in 32% yield starting from 10.8 g of PEG400 (4c) following the same procedure as described for 3a. 1 H NMR (CDCl3): δ 3.740 (4H, t, BrCH2CH2-); 3.585 (26H, m, -OCHH2-); 3.405 (4H, t, BrCH2-). 13 C NMR (CDCl3): δ 70.95; 70.33; 30.12. PEG 600 Dibromide (3d). Compound 3d was obtained in 22% yield starting from 11.2 g of PEG600 (4d) following the same procedure as described for 3a. 1 H NMR (CDCl3): δ 3.735 (4H, t, BrCH2CH2-); 3.571 (44H, m, -OCHH2-); 3.396 (4H, t, BrCH2-). 13 C NMR (CDCl3): δ 71.02; 70.40; 30.17. PEG 1000 Dibromide (3e). PBr3 (2.38 mL, 0.03 mol) was added over 3 h to a solution of PEG1000 (4e) (11.0 g, 0.01 mol) and pyridine (2.04 mL, 0.03 mol) at reflux. The reaction mixture was refluxed for 16 h, and then it was cooled and treated with 2% aqueous HCl (10 mL) and distilled water (50 mL). The mixture was extracted with CCl4 (4 50 mL) and the organic layer was dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure gave 6.67 g of 3e as a pale yellow oil (53% yield). 1 H NMR (CDCl3): δ 3.721 (4H, t, BrCH2CH2-); 3.561 (78H, m, -OCHH2-); 3.389 (4H, t, BrCH2-). 13 C NMR (CDCl3): δ 70.96; 70.34; 30.15. Langmuir 2010, 26(9), 6177–6183
Article
Preparation of Aqueous Surfactant Dispersions. A film of lipid was prepared on the inside wall of a round-bottom flask by evaporation of CHCl3 solutions containing the proper amount of N-oxide surfactant. The obtained films were stored overnight under reduced pressure (0.4 mbar); 2.5 mL of PBS buffer solution (Aldrich, 0.15 M, pH 7.4) was added to the lipid film in order to obtain a 12.5 mM lipid dispersion, and the solutions were vortex mixed and then freeze-thawed six times from liquid nitrogen to 40 C. Dispersions were then extruded (10 times) through a 100 nm polycarbonate membrane (Whatman Nucleopore). The extrusions were carried out well above the transition temperature of mixed liposomes, using a 2.5 mL extruder (Lipex Biomembranes, Vancouver, CA). Determination of the Critical Aggregation Concentrations, CACs, by Fluorescence Spectroscopy Measurements.
The CAC of surfactants 1a-1e was measured at 30 C by a procedure that exploits the variation of the intensity of the vibronic fine structure in pyrene monomer fluorescence upon association with micellar aggregates.19,20 In fact, the intensity of the vibronic bands is perturbed by variations of the solvent polarity, therefore the variation of the ratio of vibronic band intensity (I3/I1;the intensity of the third and first vibronic peaks of pyrene at 380 and 370 nm, respectively) as a function of surfactant concentration is used as an excellent method for measuring the CAC (corresponding to the flex point in the plot). This method is unaffected by the very slow reorganization at the air/water interface typical of GSs, further, it also provides information on the polarity of the interfacial region of aggregates;3,7 in particular, lower values of the ratio correspond to a higher polarity sensed by pyrene and hence to a major water penetration.19 Aqueous unextruded solutions (3 mL) of each N-oxide based GS at concentrations between 1.0 mM and 0.10 μM were added to a defined amount of pyrene to obtain a 1.1 μM final concentration of pyrene (prepared from 50 μL of an ethanol solution of 67.4 μM pyrene dried by a nitrogen flux). The solutions were kept above 37 C, with stirring, for 12 h. The fluorescence measurements were performed at room temperature on a FluoroMax-4 HoribaJobinYvon spectrofluorimeter. Emission spectra of the solutions were acquired in the range 350-450 nm (λexc = 335 nm).
Determination of the Aggregate Size by Dynamic Light Scattering (DLS) Measurements. N-Oxide based GS solutions used for size characterization were 0.75 mM in 7.5 mM PBS buffer. The size and size distribution of the aggregates were measured on unextruded samples as a function of the temperature using a Malvern NanoZetaSizer spectrometer, equipped with a 5 mW HeNe laser (wavelength λ = 632.8 nm) and a digital logarithmic correlator. The normalized intensity autocorrelation functions were measured at an angle of 173. The autocorrelation functions were analyzed by using the Contin algorithm.21,22 The decay times were used to obtain the distribution of the diffusion coefficients D of the particles, further converted into a distribution of the effective hydrodynamic radii, RH, using the StokesEinstein relationship RH = kBT/6πηD, where kBT is the thermal energy and η the solvent viscosity. The values of the radii reported here correspond to the average on the “intensity weighted” distribution.21,22
Determination of the Aggregate ζ Potential by Laser Doppler Velocimetry Measurements. Unextruded samples of
N-oxide based GS used in ζ potential measurements were 0.15 mM in 1.5 mM PBS buffer. In these experimental conditions low voltages were applied to avoid possible artifacts due to sample damage caused by Joule heating. (19) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 9, 2039. (20) Ananthapadmanabhan, K. P.; Goddard, E. D.; Turro, N. J.; Kuo, P. L. Langmuir 1985, 1, 35. (21) Provencher, S. Comput. Phys. Commun. 1982, 27, 213–242. (22) De Vos, C.; Deriemaeker, L.; Finsy, R. Langmuir 1996, 12, 2630.
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Bordi et al. Table 1. Values of the Physico-Chemical Parameters of the Aggregates Formed by Surfactants 1a-1e 1a
1b -5
1c
The measurements of the electrophoretic mobility u to determine the ζ potential of the particles were carried out employing a laser Doppler electrophoresis technique by using the MALVERN NanoZetasizer apparatus, already described for size measurements; u was converted into the ζ potential using the Smoluchowski relation ζ = uη/ε, where η and ε are the viscosity and the permittivity of the solvent phase, respectively. Analysis of the Doppler shift in the Zetasizer Nano series is done by using phase analysis light scattering (PALS).23
Determination of the Aggregate Surface Potential by Fluorescence Spectroscopy Measurements. This was carried out on GS samples prepared as described above using a lipophilic pH-sensitive fluorophore, 4-heptadecyl-7-hydroxycoumarin (HC), following a procedure described in the literature.24 Briefly the procedure consists in the titration of HC bound to the aggregates. In fact, its pKa is correlated to the surface potential of the aggregates where it is localized. Because the undissociated and the dissociated forms of HC feature absorption maxima at different wavelengths and the fluorescence of this molecule is quenched in water, it is quite simple to follow protonation during titration by excitation fluorescence experiments without interferences from the unbound probe.24 HC containing aggregates were prepared by adding to the lipid chloroform solution the proper volume of a HC stock solution (5 10-4 M, THF) to obtain, after hydration, a final concentration of 50 μM of HC. In all samples, the molar ratio of lipids to HC was 250 to 1. The preparation of HC-containing aggregates as well as all the experiments involving HC, were performed in the dark to avoid HC photodegradation. After the extrusion 100 μL of liposome dispersion was diluted in 2.5 mL of PBS buffer (pH 7.4). We considered 1,2-dimyristoyl-sn-glycero-3-phosphatidylcoline liposomes as a neutral reference, assuming that there is no change in surface polarity (pKa 10). The fluorescence measurements were performed at room temperature on a FluoroMax-4 HoribaJobinYvon spectrofluorimeter. Fluorescence of HC was measured by scanning at the excitation wavelength between 300 and 400 nm at an emission wavelength of 450 nm (bandwidths 5 nm).
Transmission Electron Microscopy (TEM) Measurements. Samples for TEM observation were prepared by deposit-
ing 20 μL of unextruded aggregate solution onto a 300-mesh copper grid for electron microscopy covered by thin amorphous carbon film (20 nm). Samples were kept at the desired temperature in a thermostatting bath until deposition. Immediately after deposition, the excess of liquid was rapidly removed by filter paper. For negative staining, 10 mL of 2% aqueous phosphotungstic acid solution (pH-adjusted to 7.3 using 1 N NaOH) were added before samples were completely dried. Selected samples were prepared with a small amount of CsCl (CsCl final concentration 10 mM) added to the PBS buffer used in the preparation of aggregate solutions and adjusting the PBS (23) Tscharnuter, W. W. Appl. Opt. 2001, 40, 3995. (24) Zuidan, N. J.; Barenholz, Y. Biochim. Biophys. Acta 1997, 1329, 211.
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-5
1d -5
1e
9.0 10 4.2 10 5.2 10 9.0 10 1.6 10-5 CAC (M) c c diameter (nm)b at 30 C 730 ( 60 480 ( 100 780 ( 120 c 1100 ( 100 300 ( 30 370 ( 20 140 ( 20 diameter (nm)b at 60 C ζ potential (mV) at 30 C 29 ( 4 9(3 22 ( 5 31 ( 6 42 ( 7 ζ potential (mV) at 60 C 10 ( 8 8(3 32 ( 7 43 ( 5 66 ( 10 d 35 37 44 36 144 surface potential in PBS buffer (mV) d 50 48 47 55 228 surface potential in water (mV) pH in water 5.8 5.9 5.7 6.0 7.0 1.1 1.5 1.7 1.7 1.6 I3/I1 a Error in CAC values are estimated to be less than 10%. b The diameter corresponds to the average value calculated accorded to the intensity weighted size-distribution obtained by CONTIN algorithm. The error in size values corresponds to the standard deviation of the size distribution. c Polydisperse population with not measurable large size aggregates (>1 μm hydrodynamic diameter). d Error in determination is (5 mV. a
-5
concentration to have the same final ionic strength of the samples without CsCl. This element, entrapped in the aqueous domain of the aggregates, produces a strong contrast due to its relatively high atomic number.25 Images obtained with this procedure, where the aggregates are stained from the inside, are complemental to those obtained by negative staining. TEM measurements were carried out by means of a Zeiss 902 microscope (Carl Zeiss, Jena, Germany) operating at 80 kV, equipped with an electron energy loss filter (EFTEM) and with the capability of performing electron energy loss spectra (EELS). Images were acquired by a digital charge-coupled device camera, model Proscan (Proscan Elektronische Systeme, Lagerlechfeld, Germany) HSC2 (1024 3 1024 pixels), thermostated by a Peltier cooler. Image analysis was carried out by a digital analyzer SIS 3.0, which allowed us to obtain elemental (in our case, Cs) maps using the two-windows method (50), to enhance the contrast and sharpness of the images and to perform morphological analysis.
Results and Discussion The results obtained by the physicochemical characterization of the aggregates formed by the new N-oxide based GSs are reported in Table 1. The CAC of surfactants 1a-1e was measured at 30 C by a known procedure19,20 that exploits the variation of the intensity of the vibronic fine structure of pyrene monomer fluorescence upon association with micellar aggregates, and also provides information on the polarity of the interfacial region of aggregates as explained in the experimental part. All the GSs feature low CACs; the sigmoids obtained by plotting the ratio of the intensity of the third and first vibronic peaks of pyrene, I3/I1, versus the concentration of surfactant are rather broad, probably because of the tendency, very common in GSs surfactants, to form premicellar aggregates.26,27 In Figure 1 we report, as an example, the plot relative to 1c (the other plots are available as SI). The values reported in Table 1 are relative to the concentrations at the flex points. The values of CACs relative to GSs 1a-1c follow an expected trend, because the CAC decreases in correspondence of an increase of the hydrophobic portion (in the comparison of 1a with 1b), whereas it slightly increases by increasing the hydrophilic portion (in the comparison of 1b with 1c). The increase of both the hydrophilic and hydrophobic portions in 1d and 1e with the possible occurrence of a large extent of cation and H3Oþ complexation in 1e, due to its longer spacer, make the rationalization of the observed values difficult. Table 1 also lists the limiting values of the I3/I1 ratio relative to concentrations of GS well above the CAC, where the fluorescent probe is fully partitioned in the aggregates. Since pyrene solubilizes (25) Bordi, F.; Cametti, C.; Diociaiuti, M.; Sennato, S. Biophys. J. 2006, 9, 1513. (26) Frindi, M.; Michels, B.; Levy, H.; Zana, R. Langmuir 1994, 10, 1140. (27) Zana, R.; Benrraou, M.; Rueff, R. Langmuir 1991, 7, 1072.
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Figure 1. Plot of the ratio of the intensity of the third and first vibronic peaks of pyrene, I3/I1, versus the concentration of GS 1c.
preferentially in the hydrophobic region of the aggregates, the variations of I3/I1 reflect the changes in the penetration of water in the aggregates and then in the packing of the hydrophobic chains. The smallest value obtained in the case of 1a would suggest a higher penetration of water in the hydrophobic region of 1a aggregates. DLS and ζ potential measurements reported in Table 1 were carried out, at both 30 and 60 C, on unextruded samples, whereas surface potential measurements were carried out on extruded samples for avoiding possible disturbance due to the scattering connected with the presence of large aggregates. However, DLS measurements on extruded samples showed that the obtained aggregates tend to fuse in large aggregates, as observed in the unextruded samples, in relatively short times. As a general observation all the aggregates are characterized by positive surface and ζ potential values despite the zwitterionic nature of the polar head groups, thus suggesting interaction of the negatively charged oxygen of N-oxide head groups with cations (included H3Oþ). The GSs characterized by the shortest spacer, i.e., 1a and 1b, formed very large aggregates at both the temperatures. Nevertheless, the size of the aggregates of 1a at 30 and 60 C was significantly lower than that of 1b and thus measurable, though with low accuracy. At both temperatures, aggregates of 1b showed larger size and were very large and irregularly shaped, so that their average radius could not be reliably assessed by DLS. In fact, for particles whose size is comparable to the wavelength of the light employed, the intraparticle interference strongly affects the values of the radii obtained from the measurement. This effect should be taken into account trough a properly determined “ form factor”;28 however, for irregularly shaped and “fluffy” aggregates the form factor cannot, in general, be calculated.29 The increase of the size of the aggregates of 1a at higher temperature suggests an aggregation driven by hydrophobic interactions. A different behavior was observed for the aggregates formed by 1c, 1d, and 1e, whose size was smaller at 60 C compared to that observed at 30 C. As an example of this behavior in Figure 2 we report the average hydrodynamic diameter of the aggregates of 1a (panel A) and 1e (panel B) in the range of temperature from 30 to 70 C. In the case of 1a, we can observe a monotonous and rather small, although appreciable, increase of the average size, and a broadening of the size distribution as a function of increasing tempera(28) Dhont, J. K. G. An Introduction to Dynamics of Colloids; Elsevier: Amsterdam, 1996. (29) Bordi, F.; Cametti, C.; Sennato, S.; Truzzolillo, Phys. Rev. E 2007, 76, 61403 and literature cited therein.
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Figure 2. Plot of the average hydrodynamic diameter of the aggregates formed by of N-oxide based GS 1a (panel A) and 1e (panel B) in aqueous solution as a function of temperature. The insets show the distribution of hydrodynamic diameter calculated from the CONTIN algorithm at two selected temperatures, 30 (empty bars) and 60 C (full bars). For 1a (panel A), the increase of temperature induced a continuous and small, although appreciable, increase of the average size, and a broadening of the size distribution. Conversely, for 1e, it induced a sharp and abrupt change of the diameter and narrowing of size distribution.
Figure 3. TEM image of the aggregates formed by GS 1a at 30 C. Negative staining shows the indented contour of the aggregates that is suggestive of a complex structure, with bulges and bumps protruding from the surface. Bar represents 100 nm.
ture whereas in the case of 1e we observe a sharp change of the diameter in a narrow range of temperature (at ∼43 C), and significant narrowing of the size distribution. Such behavior suggests the occurrence of a phase transition. Changes observed in function of temperature were reversible in all cases. Samples were stable over at least 2 weeks as monitored by DLS and ζ potential measurements. Interestingly, if we compare the values of ζ potential obtained at the two temperatures with the size of the corresponding aggregates, we observe that the change from a higher to a lower positive ζ potential corresponds, for the same sample, to the change from smaller to larger aggregates. Higher values of ζ potential are due to a higher extent of interaction of the N-oxide DOI: 10.1021/la1005067
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Figure 4. TEM images of the aggregates formed by GS 1a at 30 C. Contrast is due to the cesium salt added in the preparation. The two images represent a sequence. In panel A the interior of the aggregate appears crowded by irregularly shaped blobs, that resemble the structure of a pomegranate, with rather large grains packed closely, and wrapped up in a structure of thin, sinuous sheets. Because the brighter areas are due to a lower concentration of cesium and hence represent the more hydrophobic domains, these thin sheets could be interpreted as surfactant layers, and the whole brain-like convoluted structure as due to the presence of extended, folded multilayers. Panel B reports the image of the same particle taken after a prolonged exposition to the electron beam thus showing the damaging of the aggregate and the cesium leak due to the prolonged exposition. Here the internal region appears more confused because some blobs exploded, and a halo of cesium appears around the aggregate as diffusing away. Brighter areas are the regions where blobs lost their original structure due to leakage of their aqueous (and cesium) content. Bars represent 100 nm.
head groups with the cations, in samples buffered with PBS, and to a higher extent of interaction with H3Oþ ions, in samples devoid of PBS, therefore it results that for the same GS, in correspondence of a more charged interface, we have smaller aggregates (or viceversa). To gain more insight into the structure of the aggregates formed by the different surfactants we carried out a morphological study by electron microscopy. Figure 3 shows a typical image of the large particles formed in aqueous solution by GS 1a at 30 C. 6182 DOI: 10.1021/la1005067
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Figure 5. TEM images of the aggregates formed by 1e at 30 C, at different magnification. Contrast is due to the cesium salt added in the preparation and samples are not negatively stained. Bars represent 250 nm. Rather large and irregularly shaped particles appear to be formed by smaller (∼50-100 nm in diameter) and more regular aggregates. At greater magnification (insets 1 and 2 of panel B) these globules appear in turn to be made of small quasispherical particles, probably micelles, with a diameter of ∼3-4 nm. In larger assemblies these particles are ordered in a para-crystalline assembly (inset 1), being organized in a regular lattice whose characteristic length,calculated by a fast Fourier transform of the images, is 3.6 nm. In the insets, bar is 40 nm.
The negative staining permits to distinguish the indented contour of the aggregates, that is suggestive of a complex structure, with bulges and bumps protruding from the surface, however this staining technique can not provide any direct information on the inner morphology of the particles. Cs salts can be effectively employed to enhance the TEM contrast of lipid assemblies formed in an aqueous medium and devoid of inherent contrast.25 Therefore, we prepared the aggregates in the presence of a small amount of CsCl that, without affecting significantly the structure of the aggregates, enhanced their inner contrast by staining their hydrophilic\water compartments, due to the relatively high atomic number of Cs. In panel A of Figure 4, the Cs contrast reveals a complex internal morphology, with a brain-like convoluted appearance that suggests a structure of extended, folded multilayers. In panel B of Figure 4, we report the image of the same Langmuir 2010, 26(9), 6177–6183
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particle as in panel A taken after a prolonged exposition to the electron beam. Because of the heating of the beam, the internal structure of the particle has rapidly changed. A dark halo appears around the particle and brighter zones appear inside the particle. The ESI-TEM elemental analysis clearly demonstrated (images not shown) that the dark halo is due to the Cs that leaked out of the particle. This “destructive” experiment clearly supported the view that the cesium salt was entrapped in water compartments segregated within the lipid matrix of the particle. In fact, when the internal architectures containing water and cesium salt were progressively destroyed by the heat of the beam, the cesium was expelled from the particle, transported by vaporizing water. Conversely, the large aggregates formed at low temperature by 1e (Figure 5), even at a relatively low magnification, clearly appear as made of smaller spherical subunits, with a diameter of 50-100 nm. At greater magnification (inset of panel B) it is evident the “pomegranate structure” of these spherical subunits that are made of small aggregates, probably micelles, with a diameter of approximately 3-4 nm. In the largest of the spherical subunits these small particles (3-4 nm) are organized in a paracrystalline architecture (inset 1 of Figure 5) that feature a length of 3.6 nm (calculated by a Fastr Fourier Transform of the image). Figure 6 shows the aggregates of 1e deposited after incubation at 60 C; here we can observe only isolated globules smaller than 100 nm. On the basis of these results we suggest that, due to a different hydrophilic/hydrophobic balance of their molecular structure, the GSs 1a and 1b form aggregates that differ from those formed by 1c, 1d, and 1e. 1a and 1b spontaneously form large aggregates characterized by a low curvature, probably extended planar bilayers where head groups are more or less packed depending on the hydrophobic penalty. In particular, 1a, characterized by shorter hydrophobic tails with respect to 1b, at 30 C forms aggregates probably with a modest headgroup packing and a consequent larger extent of water penetration (as shown by I3/I1 value) due to a less relevant “hydrophobic disadvantage” with respect to aggregates of 1b at the same temperature; the minor extent of headgroup packing allows a higher extent of their protonation, as suggested by the measured value of the ζ potential, which at 30 C is significantly higher for 1a than for 1b. At 60 C, when the penalty in term of reduced entropy of the solvent becomes too high also for the short tailed 1a and the control on aggregation by tail to tail interactions becomes more important, the formation of highly packed aggregates characterized by shorter headgroup distances and scarce protonation, appears favored for both these GS. Conversely, 1c, 1d, and 1e form small aggregates, probably micelles, hierarchically organized in spherical units that form very large cluster at 30 C. The increase of temperature, from 30 to 60 C, induces the decrease of particle size, a significant narrowing of the size distribution, and increase of the values of the electrokinetic potential at the surface of shear between the charged surface of the aggregates and the bulk, the ζ potential, increase. In fact, the increase of temperature breaks the large clusters of aggregates into the spherical units that become stabilized by a higher extent of N-oxide protonation due to the higher ionization of water. The different hydrophilic/hydrophobic balance of the surfactant molecular structure, that in the aggregating process entails the competing effects of the reduction of conformational entropy of the spacer and of the increase of water entropy, is responsible of the differences observed between 1c, 1d, and 1e. The values of surface potential were obtained on extruded aggregates by measuring the effective pH at the interface through the dissociation constant of a lipophilic fluorescent probe, HC. Langmuir 2010, 26(9), 6177–6183
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Figure 6. TEM images of the aggregates of 1e at 60 C. Contrast is due to the cesium salt added in the preparation and the sample is not negatively stained. At 60 C the large and irregularly shaped aggregates observed at 30 C (Figure 5) disassemble in their smaller components. Bar represents 100 nm.
The positive values obtained both in the presence and in the absence of buffer are further evidence of protonation at the interface; interestingly, the extruded aggregates formed by 1a, 1b, 1c, and 1d show a similar extent of protonation whereas the aggregates of 1e are definitely more charged. This strong difference is confirmed by the pH of the bulk that is higher in the case of 1e vesicle suspension, H3Oþ ions being concentrated on the surface of the aggregates. Our hypothesis is that the longer and “entropically more flexible” hydrophilic spacer of 1e can more easily form loops that stabilize the charged head groups. These new surfactants that combine the features of GSs and N-oxides can be exploited in different fields upon modulation of their molecular structure. Those characterized by a low extent of protonation (1a and 1b) could find application in the cleaning and protection of stonewalls, whereas the high extent of protonation of 1e could be exploited in the formulation of cationic liposomes for biomedical applications. Further, the ability of the large PEG type portion of the GS to favor cation/N-oxide interactions suggests the possibility to associate to the aggregate surface various kind of metal cations, Lewis acids etc., to be used in water remediation and/or as catalysts in various reactions.
Conclusions We have synthesized new double alkyl chain N-oxide based GSs differing in the length of the alkyl chain and/or of the polyoxyethylenic spacer. The different balance of the hydrophilic and hydrophobic portions of the molecule were shown to ascribe to the various GSs very different aggregating, morphological and electrical features that can be exploited in different fields of application. Acknowledgment. The authors thank for financial support the project PON SAPAB and the Dipartimento di Progettazione Molecolare of CNR. Supporting Information Available: Figures S1-S4, showing plots of the ratio of the intensity of the third and first vibronic peaks of pyrene, I3/I1, versus the concentration of GS, for the determination of CAC of surfactants 1a, 1b, 1d, and 1e. This material is available free of charge via the Internet at http://pubs.acs.org. DOI: 10.1021/la1005067
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