synthesis and physicochemical characterization of new twin-tailed n ...

DOI: 10.1021/la1005067 6177Langmuir 2010, 26(9), 6177–6183 Published on Web 03/31/2010

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, 00185Roma, Italy, ‡Dipartimento di Chimica, IngegneriaChimica eMateriali, Universit�a degli Studi deL’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 MetodologieChimiche;Sezione Meccanismi di Reazione and Dipartimento di Chimica, Universit�a degli Studi di Roma “LaSapienza”, P. le AldoMoro 5, 00185Roma, Italy, )Centro di EccellenzaMateriali Innovativi Nanostrutturali perApplicazioni 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-oxidemonovalent surfactants joined by a PEG spacer of different length (from 3 to 21 ethylene glycol units), thus combiningthe 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 theaggregates. Despite the zwitterionic nature of the polar head groups, all the aggregates are characterized by positivepotential thus suggesting protonation at the interface; however, the extent of protonation was shown to strongly dependon the length of the alkyl chain and of the spacer.

Introduction

Gemini surfactants1,2 (GSs) are amphiphilic molecules that con-tain two head groups and two aliphatic chains, linkedby a rigid3-6

or flexible7,8 spacer. Their molecular structure confers them verypeculiar physicochemical properties compared to the correspond-ing monovalent surfactants. In fact, they typically show highlysuperior surfactant properties with respect to the correspondingconventional amphiphiles; for example, surface activity can beincreased 1000-fold. Moreover, GSs are characterized by lowercritical micellar concentration (cmc) values, higher solubilizationpower, and hydrotropy with respect to the corresponding mon-ovalent surfactants. The higher surface activity of GSs is advan-tageous for their applications in the industry for detergency andemulsification and involves the use of smaller amounts of rawmaterial for synthesis and the handling of less manufacturing andbyproduct, thus ending in a minor environmental impact.9 Allthese advantagesmake themof special interest also for biomedicalapplications, where they have been investigated as drug delivery

systems10 and DNA carriers in transfection studies.11 Because ofthese features, though the family of GSs is relatively young(actually, they were first reported ∼40 years ago,12 but a largeinterest for these surfactants has spread ∼20 years later), therealready are a large number of species for a whole range ofapplications. So far over 10000 international patents on GSshave been filled, and investigations on many different applica-tions are currently being reported. The manipulation of the basicstructure of gemini can give an almost unlimited number ofpotential molecules, thus allowing extensive structure-activitystudies aimed at identifying structural features necessary for thesuccessful exploitation of GSs.

Here we report the preparation of the new GSs (Scheme 1),composed of two (from 7 to 17 methylenic units) N-oxidemonovalent surfactants bearing two alkyl chains and joined bya PEG spacer of different length (from 3 to 21 ethylene glycolunits), and the physicochemical characterization of the aggregatesthey form inwater.N-Oxide surfactants are in general biodegrad-able, show a low-to-moderate toxicity, and show good antiox-idant13 and antimicrobial activity14 (both depending on the alkylchain length). N-oxide surfactants are used in many cleaningformulations, in liquid bleach products, as antistatic agent intextile industry, as foam stabilizer in the rubber industry, aspolymerization catalysts in polymer industry, in anticorrosioncompositions, as lime soap dispersants, and as antibacterialagents in deodorant bars due to their compatible synergistic effectand environment friendly nature.15 The absence of counterions in

*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 thetime of publication, G.C.’s telephones were not working, as the Chemistrybuilding 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.

(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) Bukowsk�y, M.; Mlinar�cik, D.; Ondra�ckova, 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.

6178 DOI: 10.1021/la1005067 Langmuir 2010, 26(9), 6177–6183

Article Bordi et al.

N-oxide surfactants make themparticularly interesting also in thefield of cultural heritage (cleaning and protection of stonewall), infact, the presence of salts can modify the capability of water toseep into the stone, causing serious damage to themonuments as aconsequence of freeze and thaw cycles.16 Furthermore, they areinvestigated in pH controlled DNA condensation.17 Combiningthe properties of N-oxide surfactants with a GS type structurecould further develop the potential of this compounds, extendingthe areas of their application or improving their performances.18

Experimental Section

Materials. Phosphorus tribromide (PBr3), pyridine, tetraethy-lene glycol (TEG), poly(ethylene) glycol 400 (PEG 400), poly-(ethylene) glycol 600 (PEG 600), poly(ethylene) glycol 1000 (PEG1000), di-n-octylamine, di-n-decylamine, di-n-octadecylamine,and manganese oxide (MnO2) were purchased from Sigma-Aldrich and used without further purification. Solvents: HCl,Na2SO4,Na2CO3, andH2O2 40%m/vwere purchased fromCarloErba Reagenti.

Bis(di-n-octylamine)bis(ethoxyethyl)Ether-N-oxide (C10)2-NþO--TEG-NþO-(C10)2 (1a). H2O2, 0.33 mL (40% m/v,3.9 mmol), was added dropwise with continuous stirring to asolution 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 toroom temperature and the excess of H2O2 decomposed withMnO2. The solid MnO2 was removed by filtration, washed withethanol and the solventwas removed under reduced pressure. Theresiduewasdissolved indiethyl ether and the solventwas removedunder reduced pressure three times giving 1.1 g (90%) of crudeproduct 1a as brownish oil. The product was purified by chro-matography on silica gel eluting with chloroform-methanol(95:5, v/v) to give 750 mg of 1a as yellow oil (71% yield).

1H 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).13C 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)2-NþO--TEG-NþO-(C10)2 (1b). 1b was obtained in 20%

yield as yellow oil starting from 2b (1.0 g) following the sameprocedure described for 1a.

1HNMR (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).13C 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-tained starting from 1.0 g of 2c following the same proceduredescribed for 1a. The crude product 1c was dissolved in CH2Cl2(5 mL), added dropwise with diethyl ether (200 mL) and kept infreezer for 24h.The precipitatewas collected by filtration,washedwith diethyl ether, and dried under vacuum to give 300mgof 1c asa yellow oil (30% yield).

1H 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).13C 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)2NþO--PEG600-NþO- (C18)2 (1d). The crude compound1d was obtained starting from 1.0 g of 2d following the sameprocedure described for 1a. Using the same method of purifica-tion described for 1c, 230 mg of 1d as pale yellow crystals wereobtained (23% yield).


(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).13C 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)2-N

þO

--PEG1000-NþO

-(C18)2 (1e). The crude compound1e was obtained starting from 1.0 g of 2e following the sameprocedure described for 1a. Using the same method of purifica-tion as described for 1c, 230 mg of 1e as light yellow crystals wereobtained (22% yield).


(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).13C 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 sol-vent 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 at120 �C. Themixture was cooled to room temperature, added with10 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 compound2a (2.4 g, 78% yield).

1H NMR (CDCl3): δ 3.526 (12H, m, -CH2OCH2-); 2.569(4H, t, (C10)2N

þO-CH2-); 2.358 (8H, t, -CH2NþO-TEG-);


0.802 (12H, t, -CH3).13C 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). Com-pound 2b was obtained in 80% yield starting from 3a and di-n-decylamine following the same procedure described for 2a.


þO-CH2-); 2.637 (8H, m, -CH2NþO-TEG-);


0.827 (12H, t, -CH3).

Scheme 1. Synthetic Pattern for the Preparation of GSs 1a-1ea

aKey: (i) PBr3, pyridine; (ii) 2a, di-n-octylamine, 2b, 2c di-n-decyla-mine, 2d, 2e di-n-octadecylamine, Na2CO3, 120 �C; (iii) H2O2, absoluteethanol, reflux.

(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.

DOI: 10.1021/la1005067 6179Langmuir 2010, 26(9), 6177–6183

Bordi et al. Article

13C 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.

PEG 400 N,N-Di-n-decylamine (C10)2N-PEG400-N(C10)2 (2c). Compound 2cwas obtained in 94% yield startingfrom 3c and di-n-decylamine following the same proceduredescribed for 2a.


þO-CH2-); 2.347 (8H, t, -CH2NþO-TEG-);

1.291 (8H,bs,-CH2CH2NþO-TEG-); 1.177 (56H,m,-(CH2)7-);

0.795 (12H, t, -CH3).13C 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-PEG600-N(C18)2 (2d). Compound 2dwas obtained in 80% yield startingfrom 3d and di-n-octadecylamine following the same proceduredescribed for 2a.


þO-CH2-); 2.396 (8H, t,-CH2NþO-TEG); 1.423

(8H, bs, -CH2CH2NþO-TEG); 1.215 (120H, m, -(CH2)15-);

0.837 (12H, t, -CH3).13C 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.

PEG1000N,N-Di-n-octadecylamine (C18)2N-PEG1000-N(C18)2 (2e). Compound 2ewas obtained in 75% yield startingfrom 3d and di-n-octadecylamine following the same proceduredescribed for 2a.


þO-CH2-); 2.813 (8H, m, -CH2NþO-TEG);

1.637(8H,bs,-CH2CH2NþO-TEG);1.186(120H,m,-(CH2)15-);

0.807 (12H, t, -CH3).13C 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 (0.02mol) of PBr3 was cooled under nitrogen in a round-bottom flaskto 0 �C using an ice bath. Amixture of TEG (4a) (5.6 g, 0.03 mol)and pyridine (0.81mL, 0.01mol) was added to the solution slowlyunder stirring. The reactionmixturewas allowed towarm to roomtemperature and to react for 8 h. The solution was added withwater (50mL), neutralizedwithHCl and extractedwithCCl4 (3�50 mL) and the organic layer was dried over anhydrous Na2SO4.Removal of the solvent under reduced pressure gave compound4.1 g of 2a (42% yield).

1HNMR(CDCl3):δ 3.749 (4H, t,BrCH2CH2-); 3.610 (8H,m,-OCHH2-); 3.413 (4H, t, BrCH2-).

13C NMR (CDCl3): δ 70.96; 70.42; 70.30; 30.27.

PEG 400 Dibromide (3c). Compound 3c was obtained in32%yield starting from10.8 g of PEG400 (4c) following the sameprocedure as described for 3a.

1H NMR (CDCl3): δ 3.740 (4H, t, BrCH2CH2-); 3.585 (26H,m, -OCHH2-); 3.405 (4H, t, BrCH2-).

13C NMR (CDCl3): δ 70.95; 70.33; 30.12.

PEG 600 Dibromide (3d). Compound 3d was obtained in22%yield starting from11.2 g of PEG600 (4d) following the sameprocedure as described for 3a.

1HNMR(CDCl3): δ 3.735 (4H, t, BrCH2CH2-); 3.571 (44H,m,-OCHH2-); 3.396 (4H, t, BrCH2-).

13C NMR (CDCl3): δ 71.02; 70.40; 30.17.

PEG 1000 Dibromide (3e). PBr3 (2.38 mL, 0.03 mol) wasadded 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 mixturewas refluxed for 16 h, and then it was cooled and treated with 2%aqueous HCl (10 mL) and distilled water (50 mL). The mix-ture was extracted with CCl4 (4 � 50 mL) and the organic layerwas dried over anhydrous Na2SO4. Removal of the solventunder reduced pressure gave 6.67 g of 3e as a pale yellow oil(53% yield).

1H NMR (CDCl3): δ 3.721 (4H, t, BrCH2CH2-); 3.561 (78H,m, -OCHH2-); 3.389 (4H, t, BrCH2-).

13C NMR (CDCl3): δ 70.96; 70.34; 30.15.

Preparation of Aqueous Surfactant Dispersions. A film oflipid was prepared on the inside wall of a round-bottom flask byevaporation of CHCl3 solutions containing the proper amount ofN-oxide surfactant. The obtained films were stored overnightunder reduced pressure (0.4mbar); 2.5 mL of PBS buffer solution(Aldrich, 0.15 M, pH 7.4) was added to the lipid film in order toobtain a 12.5 mM lipid dispersion, and the solutions were vortexmixed and then freeze-thawed six times from liquid nitrogento 40 �C. Dispersions were then extruded (10 times) through a100 nm polycarbonate membrane (Whatman Nucleopore). Theextrusions were carried out well above the transition temperatureof mixed liposomes, using a 2.5 mL extruder (Lipex Biomem-branes, Vancouver, CA).

Determination of the Critical Aggregation Concentra-

tions, CACs, by Fluorescence SpectroscopyMeasurements.The CAC of surfactants 1a-1e was measured at 30 �C by aprocedure that exploits the variation of the intensity of thevibronic fine structure in pyrene monomer fluorescence uponassociation with micellar aggregates.19,20 In fact, the intensity ofthe vibronic bands is perturbed by variations of the solventpolarity, therefore the variation of the ratio of vibronic bandintensity (I3/I1;the intensity of the third and first vibronic peaksof pyrene at 380 and 370 nm, respectively) as a function ofsurfactant concentration is used as an excellent method formeasuring the CAC (corresponding to the flex point in the plot).This method is unaffected by the very slow reorganization at theair/water interface typical of GSs, further, it also provides in-formation on the polarity of the interfacial regionof aggregates;3,7

in particular, lower values of the ratio correspond to a higherpolarity sensed by pyrene and hence to a major water penetra-tion.19

Aqueous unextruded solutions (3 mL) of each N-oxide basedGSat concentrations between 1.0mMand 0.10μMwere added toa defined amount of pyrene toobtain a 1.1μMfinal concentrationof pyrene (prepared from 50 μL of an ethanol solution of 67.4 μMpyrene dried by a nitrogen flux). The solutions were kept above37 �C,with stirring, for 12 h.The fluorescencemeasurementswereperformed at room temperature on a FluoroMax-4 HoribaJobi-nYvon spectrofluorimeter. Emission spectra of the solutions wereacquired in the range 350-450 nm (λexc = 335 nm).

Determination of the Aggregate Size by Dynamic Light

Scattering (DLS)Measurements.N-Oxide basedGS solutionsused for size characterization were 0.75 mM in 7.5 mM PBSbuffer. The size and size distribution of the aggregates weremeasuredonunextruded samples as a function of the temperatureusing a Malvern NanoZetaSizer spectrometer, equipped with a5 mW HeNe laser (wavelength λ = 632.8 nm) and a digitallogarithmic correlator. The normalized intensity autocorrelationfunctions were measured at an angle of 173�. The autocorrelationfunctions were analyzed by using the Contin algorithm.21,22 Thedecay times were used to obtain the distribution of the diffusioncoefficientsDof the particles, further converted intoadistributionof the effective hydrodynamic radii, RH, using the Stokes-Einstein relationship RH = kBT/6πηD, where kBT is the thermalenergy and η the solvent viscosity. The values of the radii reportedhere correspond to the average on the “intensity weighted”distribution.21,22

Determination of the Aggregate ζ Potential by Laser

Doppler Velocimetry Measurements. Unextruded samples ofN-oxide based GS used in ζ potential measurements were 0.15mM in 1.5 mM PBS buffer. In these experimental conditions lowvoltages were applied to avoid possible artifacts due to sampledamage 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.

6180 DOI: 10.1021/la1005067 Langmuir 2010, 26(9), 6177–6183


The measurements of the electrophoretic mobility u to deter-mine the ζ potential of the particles were carried out employing alaserDoppler electrophoresis technique by using theMALVERNNanoZetasizer apparatus, already described for size measure-ments; u was converted into the ζ potential using the Smolu-chowski relation ζ= uη/ε, where η and ε are the viscosity and thepermittivity of the solvent phase, respectively. Analysis of theDoppler shift in the Zetasizer Nano series is done by using phaseanalysis light scattering (PALS).23

Determination of the Aggregate Surface Potential by

Fluorescence Spectroscopy Measurements. This was carriedout onGS samples prepared as described above using a lipophilicpH-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 tothe aggregates. In fact, its pKa is correlated to the surface potentialof the aggregates where it is localized. Because the undissociatedand the dissociated forms of HC feature absorption maxima atdifferent wavelengths and the fluorescence of this molecule isquenched in water, it is quite simple to follow protonation duringtitration by excitation fluorescence experiments without interfer-ences from the unbound probe.24

HC containing aggregates were prepared by adding to the lipidchloroform solution theproper volumeof aHCstock solution (5�10-4 M, THF) to obtain, after hydration, a final concentration of50 μM of HC. In all samples, the molar ratio of lipids to HC was250 to 1. The preparation of HC-containing aggregates as well asall the experiments involving HC, were performed in the dark toavoid HC photodegradation. After the extrusion 100 μL of lipo-some dispersion was diluted in 2.5 mL of PBS buffer (pH 7.4). Weconsidered 1,2-dimyristoyl-sn-glycero-3-phosphatidylcoline lipo-somes as a neutral reference, assuming that there is no change insurface polarity (pKa 10). The fluorescence measurements wereperformed at room temperature on a FluoroMax-4 HoribaJobi-nYvon spectrofluorimeter. Fluorescence of HC was measured byscanning at the excitation wavelength between 300 and 400 nm atan emission wavelength of 450 nm (bandwidths 5 nm).

Transmission Electron Microscopy (TEM) Measure-

ments. Samples for TEM observation were prepared by deposit-ing 20 μL of unextruded aggregate solution onto a 300-meshcopper grid for electron microscopy covered by thin amorphouscarbon film (20 nm).

Samples were kept at the desired temperature in a thermostat-ting bath until deposition. Immediately after deposition, theexcess of liquid was rapidly removed by filter paper. For negativestaining, 10 mL of 2% aqueous phosphotungstic acid solution(pH-adjusted to 7.3 using 1NNaOH) were added before sampleswere completely dried.

Selected samples were prepared with a small amount of CsCl(CsCl final concentration 10mM) added to the PBSbuffer used inthe preparation of aggregate solutions and adjusting the PBS

concentration to have the same final ionic strength of the sampleswithout CsCl. This element, entrapped in the aqueous domain ofthe aggregates, produces a strong contrast due to its relativelyhigh atomic number.25 Images obtained with this procedure,where the aggregates are stained from the inside, are comple-mental to those obtained by negative staining.

TEM measurements were carried out by means of a Zeiss 902microscope (Carl Zeiss, Jena, Germany) operating at 80 kV,equipped with an electron energy loss filter (EFTEM) and withthe 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 Peltiercooler. Image analysis was carried out by a digital analyzer SIS3.0, which allowed us to obtain elemental (in our case, Cs) mapsusing the two-windows method (50), to enhance the contrast andsharpness of the images and to perform morphological analysis.

Results and Discussion

The results obtained by the physicochemical characterizationof the aggregates formed by the new N-oxide based GSs arereported in Table 1.

The CAC of surfactants 1a-1e was measured at 30 �C by aknownprocedure19,20 that exploits the variationof the intensity ofthe vibronic fine structure of pyrene monomer fluorescence uponassociation with micellar aggregates, and also provides informa-tion on the polarity of the interfacial region of aggregates asexplained in the experimental part.

All the GSs feature low CACs; the sigmoids obtained byplotting the ratio of the intensity of the third and first vibronicpeaks of pyrene, I3/I1, versus the concentration of surfactant arerather broad, probably because of the tendency, very common inGSs surfactants, to form premicellar aggregates.26,27 In Figure 1we report, as an example, the plot relative to 1c (the other plots areavailable as SI). The values reported in Table 1 are relative to theconcentrations at the flex points. The values of CACs relative toGSs 1a-1c follow an expected trend, because the CAC decreasesin correspondence of an increase of the hydrophobic portion (inthe comparison of 1a with 1b), whereas it slightly increases byincreasing the hydrophilic portion (in the comparison of 1b with1c). The increase of both the hydrophilic and hydrophobicportions in 1d and 1e with the possible occurrence of a largeextent of cation and H3O

þ complexation in 1e, due to its longerspacer, make the rationalization of the observed values difficult.Table 1 also lists the limiting values of the I3/I1 ratio relative to con-centrations ofGSwell above theCAC,where the fluorescent probeis fully partitioned in the aggregates. Since pyrene solubilizes

Table 1. Values of the Physico-Chemical Parameters of the Aggregates Formed by Surfactants 1a-1e

1a 1b 1c 1d 1e

CAC (M)a 9.0 � 10-5 4.2 � 10-5 5.2 � 10-5 9.0 � 10-5 1.6 � 10-5

diameter (nm)b at 30 �C 730 ( 60 c 480 ( 100 c 780 ( 120diameter (nm)b at 60 �C 1100 ( 100 c 300 ( 30 370 ( 20 140 ( 20ζ potential (mV) at 30 �C 29 ( 4 9 ( 3 22 ( 5 31 ( 6 42 ( 7ζ potential (mV) at 60 �C 10 ( 8 8 ( 3 32 ( 7 43 ( 5 66 ( 10surface potential in PBS buffer (mV)d 35 37 44 36 144surface potential in water (mV)d 50 48 47 55 228pH in water 5.8 5.9 5.7 6.0 7.0I3/I1 1.1 1.5 1.7 1.7 1.6

aError inCACvalues are estimated to be less than 10%. bThe diameter corresponds to the average value calculated accorded to the intensityweightedsize-distribution obtained by CONTIN algorithm. The error in size values corresponds to the standard deviation of the size distribution. cPolydispersepopulation with not measurable large size aggregates (>1 μm hydrodynamic diameter). dError in determination is (5 mV.

(23) Tscharnuter, W. W. Appl. Opt. 2001, 40, 3995.(24) Zuidan, N. J.; Barenholz, Y. Biochim. Biophys. Acta 1997, 1329, 211.

(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.

DOI: 10.1021/la1005067 6181Langmuir 2010, 26(9), 6177–6183


preferentially in the hydrophobic region of the aggregates, thevariations of I3/I1 reflect the changes in the penetration of water inthe aggregates and then in the packing of the hydrophobic chains.The smallest value obtained in the case of 1awould suggest a higherpenetration of water in the hydrophobic region of 1a aggregates.

DLS and ζ potential measurements reported in Table 1 werecarried out, at both 30 and 60 �C, onunextruded samples,whereassurface potential measurements were carried out on extrudedsamples for avoiding possible disturbance due to the scatteringconnected with the presence of large aggregates. However, DLSmeasurements on extruded samples showed that the obtainedaggregates tend to fuse in large aggregates, as observed in theunextruded samples, in relatively short times. As a general obser-vation all the aggregates are characterized by positive surface andζ potential values despite the zwitterionic nature of the polar headgroups, thus suggesting interaction of the negatively chargedoxygen of N-oxide head groups with cations (included H3O

þ).The GSs characterized by the shortest spacer, i.e., 1a and 1b,

formed very large aggregates at both the temperatures. Never-theless, the size of the aggregates of 1a at 30 and 60 �C wassignificantly lower than that of 1b and thus measurable, thoughwith 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 byDLS.In fact, for particles whose size is comparable to thewavelength ofthe light employed, the intraparticle interference strongly affectsthe values of the radii obtained from themeasurement. This effectshouldbe taken into account trough a properly determined“ formfactor”;28 however, for irregularly shaped and “fluffy” aggregatesthe form factor cannot, in general, be calculated.29 The increase ofthe size of the aggregates of 1a at higher temperature suggests anaggregation driven by hydrophobic interactions. A differentbehavior was observed for the aggregates formed by 1c, 1d, and1e, whose size was smaller at 60 �C compared to that observed at30 �C. As an example of this behavior in Figure 2 we report theaverage hydrodynamic diameter of the aggregates of 1a (panel A)and 1e (panel B) in the range of temperature from 30 to 70 �C. Inthe case of 1a, we can observe a monotonous and rather small,although appreciable, increase of the average size, and a broad-ening of the size distribution as a function of increasing tempera-

ture whereas in the case of 1e we observe a sharp change of thediameter in a narrow range of temperature (at ∼43 �C), andsignificant narrowing of the size distribution. Such behaviorsuggests the occurrence of a phase transition. Changes observedin function of temperature were reversible in all cases. Sampleswere stable over at least 2 weeks as monitored by DLS and ζpotential measurements.

Interestingly, if we compare the values of ζ potential obtainedat the two temperatures with the size of the correspondingaggregates, we observe that the change from a higher to a lowerpositive ζ potential corresponds, for the same sample, to thechange from smaller to larger aggregates. Higher values of ζpotential are due to a higher extent of interaction of the N-oxide

Figure 1. Plot of the ratio of the intensity of the third and firstvibronic peaks of pyrene, I3/I1, versus the concentration of GS 1c.

Figure 2. Plot of the average hydrodynamic diameter of theaggregates formed by of N-oxide based GS 1a (panel A) and 1e

(panel B) in aqueous solution as a function of temperature. Theinsets show the distribution of hydrodynamic diameter calculatedfrom the CONTIN algorithm at two selected temperatures, 30(empty bars) and 60 �C (full bars). For 1a (panel A), the increase oftemperature induced a continuous and small, although appreci-able, increase of the average size, and a broadening of the sizedistribution. Conversely, for 1e, it induced a sharp and abruptchange of the diameter and narrowing of size distribution.

Figure 3. TEM image of the aggregates formed byGS 1a at 30 �C.Negative staining shows the indented contour of the aggregatesthat is suggestive of a complex structure, with bulges and bumpsprotruding from the surface. Bar represents 100 nm.

(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.

6182 DOI: 10.1021/la1005067 Langmuir 2010, 26(9), 6177–6183


head groups with the cations, in samples buffered with PBS, andto a higher extent of interaction with H3O

þ ions, in samplesdevoid of PBS, therefore it results that for the same GS, incorrespondence of a more charged interface, we have smalleraggregates (or viceversa).

To gain more insight into the structure of the aggregates for-med by the different surfactants we carried out a morphologicalstudy by electron microscopy. Figure 3 shows a typical image ofthe large particles formed in aqueous solution by GS 1a at 30 �C.

The negative staining permits to distinguish the indented contourof the aggregates, that is suggestive of a complex structure, withbulges and bumps protruding from the surface, however thisstaining technique can not provide any direct information on theinner morphology of the particles. Cs salts can be effectivelyemployed to enhance the TEM contrast of lipid assembliesformed in an aqueous medium and devoid of inherent contrast.25

Therefore, we prepared the aggregates in the presence of a smallamount of CsCl that, without affecting significantly the structureof the aggregates, enhanced their inner contrast by staining theirhydrophilic\water compartments, due to the relatively high atomicnumber of Cs. In panel A of Figure 4, the Cs contrast reveals acomplex internal morphology, with a brain-like convolutedappearance that suggests a structure of extended, folded multi-layers. In panel B of Figure 4, we report the image of the same

Figure 4. TEM images of the aggregates formed by GS 1a at30 �C. Contrast is due to the cesium salt added in the preparation.The two images represent a sequence. In panel A the interior of theaggregate appears crowded by irregularly shaped blobs, thatresemble the structure of a pomegranate, with rather large grainspacked closely, and wrapped up in a structure of thin, sinuoussheets. Because the brighter areas are due to a lower concentrationof cesium and hence represent the more hydrophobic domains,these thin sheets could be interpreted as surfactant layers, and thewhole brain-like convoluted structure as due to the presence ofextended, foldedmultilayers. Panel B reports the image of the sameparticle taken after a prolonged exposition to the electron beamthus showing the damaging of the aggregate and the cesium leakdue to the prolonged exposition. Here the internal region appearsmore confused because some blobs exploded, and a halo of cesiumappears around the aggregate as diffusing away. Brighter areas arethe regions where blobs lost their original structure due to leakageof their aqueous (and cesium) content. Bars represent 100 nm.

Figure 5. TEM images of the aggregates formed by 1e at 30 �C, atdifferent magnification. Contrast is due to the cesium salt added inthe preparation and samples are not negatively stained. Barsrepresent 250 nm. Rather large and irregularly shaped particlesappear to be formed by smaller (∼50-100 nm in diameter) andmore regular aggregates.At greatermagnification (insets 1 and2ofpanel B) these globules appear in turn to be made of small quasi-spherical particles, probablymicelles, with a diameter of∼3-4nm.In larger assemblies these particles are ordered in a para-crystallineassembly (inset 1), being organized in a regular lattice whosecharacteristic length,calculated by a fast Fourier transform of theimages, is 3.6 nm. In the insets, bar is 40 nm.

DOI: 10.1021/la1005067 6183Langmuir 2010, 26(9), 6177–6183


particle as in panel A taken after a prolonged exposition to theelectron beam. Because of the heating of the beam, the internalstructure of the particle has rapidly changed. A dark halo appearsaround the particle and brighter zones appear inside the particle.The ESI-TEM elemental analysis clearly demonstrated (imagesnot shown) that the dark halo is due to the Cs that leaked out ofthe particle. This “destructive” experiment clearly supported theview that the cesium salt was entrapped in water compartmentssegregated within the lipidmatrix of the particle. In fact, when theinternal architectures containing water and cesium salt wereprogressively destroyed by the heat of the beam, the cesium wasexpelled from the particle, transported by vaporizing water.

Conversely, the large aggregates formed at low temperature by1e (Figure 5), even at a relatively low magnification, clearlyappear as made of smaller spherical subunits, with a diameterof 50-100 nm. At greater magnification (inset of panel B) it isevident the “pomegranate structure” of these spherical subunitsthat are made of small aggregates, probably micelles, with adiameter of approximately 3-4 nm. In the largest of the sphericalsubunits these small particles (3-4 nm) are organized in a para-crystalline architecture (inset 1 ofFigure 5) that feature a length of3.6 nm (calculated by a Fastr Fourier Transform of the image).

Figure 6 shows the aggregates of 1e deposited after incubationat 60 �C; here we can observe only isolated globules smaller than100 nm.

On the basis of these results we suggest that, due to a differenthydrophilic/hydrophobic balance of theirmolecular structure, theGSs 1a and 1b form aggregates that differ from those formed by1c, 1d, and 1e.

1a and 1b spontaneously form large aggregates characterizedby a low curvature, probably extended planar bilayerswhere headgroups are more or less packed depending on the hydrophobicpenalty. In particular, 1a, characterized by shorter hydrophobictails with respect to 1b, at 30 �C forms aggregates probably with amodest headgroup packing and a consequent larger extent ofwater penetration (as shown by I3/I1 value) due to a less relevant“hydrophobic disadvantage” with respect to aggregates of 1b atthe same temperature; the minor extent of headgroup packingallows a higher extent of their protonation, as suggested by themeasured value of the ζ potential, which at 30 �C is significantlyhigher for 1a than for 1b. At 60 �C, when the penalty in term ofreduced entropy of the solvent becomes too high also for the shorttailed 1a and the control on aggregation by tail to tail interactionsbecomes more important, the formation of highly packed aggre-gates characterized by shorter headgroup distances and scarceprotonation, appears favored for both these GS.

Conversely, 1c, 1d, and 1e form small aggregates, probablymicelles, hierarchically organized in spherical units that form verylarge cluster at 30 �C. The increase of temperature, from 30 to60 �C, induces the decrease of particle size, a significant narrowingof the size distribution, and increase of the values of the electro-kinetic potential at the surface of shear between the chargedsurface of the aggregates and the bulk, the ζ potential, increase. Infact, the increase of temperature breaks the large clusters ofaggregates into the spherical units that become stabilized by ahigher extent ofN-oxide protonation due to the higher ionizationof water. The different hydrophilic/hydrophobic balance of thesurfactant molecular structure, that in the aggregating processentails the competing effects of the reduction of conformationalentropy of the spacer and of the increase of water entropy, isresponsible of the differences observed between 1c, 1d, and 1e.

The values of surface potential were obtained on extrudedaggregates by measuring the effective pH at the interface throughthe dissociation constant of a lipophilic fluorescent probe, HC.

The positive values obtained both in the presence and in theabsence of buffer are further evidence of protonation at theinterface; interestingly, the extruded aggregates formed by 1a,1b, 1c, and 1d show a similar extent of protonation whereas theaggregates of 1e are definitely more charged. This strong differ-ence is confirmed by the pHof the bulk that is higher in the case of1e vesicle suspension, H3O

þ ions being concentrated on thesurface of the aggregates. Our hypothesis is that the longer and“entropically more flexible” hydrophilic spacer of 1e can moreeasily form loops that stabilize the charged head groups.

These new surfactants that combine the features of GSs andN-oxides can be exploited in different fields upon modulation oftheir molecular structure. Those characterized by a low extent ofprotonation (1a and 1b) could find application in the cleaning andprotection of stonewalls, whereas the high extent of protonationof 1e could be exploited in the formulation of cationic liposomesfor biomedical applications. Further, the ability of the large PEGtype portion of the GS to favor cation/N-oxide interactionssuggests the possibility to associate to the aggregate surfacevarious kind of metal cations, Lewis acids etc., to be used inwater remediation and/or as catalysts in various reactions.

Conclusions

Wehave synthesized new double alkyl chainN-oxide basedGSsdiffering in the length of the alkyl chain and/or of the polyox-yethylenic spacer. The different balance of the hydrophilic andhydrophobic portions of themolecule were shown to ascribe to thevarious GSs very different aggregating, morphological and elec-trical features that canbe exploited in different fields of application.

Acknowledgment.The authors thank for financial support theproject PON SAPAB and the Dipartimento di ProgettazioneMolecolare of CNR.

Supporting Information Available: Figures S1-S4, show-ing plots of the ratio of the intensity of the third and firstvibronic peaks of pyrene, I3/I1, versus the concentration ofGS, for the determination of CAC of surfactants 1a, 1b, 1d,and 1e. This material is available free of charge via theInternet at http://pubs.acs.org.

Figure 6. TEM images of the aggregates of 1e at 60 �C.Contrast isdue to the cesium salt added in the preparation and the sample isnot negatively stained. At 60 �C the large and irregularly shapedaggregates observedat 30 �C (Figure 5) disassemble in their smallercomponents. Bar represents 100 nm.

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