Langmuir 1993,9, 1201-1205
1201
Synthesis and Vesicle Formation of Cationic Surfactants Based on Trisubstituted Perfluoroalkylated Thiourea H. Trabelsi, S. Szonyi,* M. Gaysinski, and A. Cambon Laboratoire de Chimie Organique du Fluor, Universitb de Nice-Sophia Antipolis, Parc Valrose, B.P. 71, F-06108 Nice Cedex 2, France
H. J. Watzke Zmtitut fiir Polymere, ETH Ziirich, Universtittitsstrasse 6, CH-8092 Zurich, Switzerland Received May 19,1992. In Final Form: February 3,1993 New perfluoroalkyldouble-chainsurfactantswere synthesized based on trisubstituted perfluoroalkylated thiourea. These amphiphiles have amodular structure consisting of hydrophobictails,thioureaas connector, alkyl spacer, and ammonium head group. The modular organization allows an independent variation of the key features in the amphiphilic structure. The synthesis strategy was based on the condensation reaction of 2-(F-alkyl)ethyl isothiocyanates with 3-(dimethylamino)-N-(2-hydroxy-2-F-alkyl)ethyl)alkylamines and subsequentquaternization by methyl iodide to produce the trisubstituted perfluoroalkylated thiourea. The two reaction steps proceed with high yieIds. The surface activity properties of the new amphiphiles were studied and the ability to self-assemble into bilayer vesicles was determined by freeze fracture electron microscopy and quasi-elasticlight scattering. Introduction In our laboratory, we have been exploring improved methods for the synthesis of perfluoroalkylated surfactants with the goal of making these materials readily accessible for basic research and surfactant technology applications. Perfluoroalkyl amphiphiles have properties that set them apart from more common hydrocarbon surfactants, in particular greater hydrophobicity, constrained conformational states of the perfluoroalkyl chains, and chemical inertness. We have been particularly interested in doublechain perfluoroalkylated surfactants which exhibit vesicle formation. Under suitable conditions many lipids or synthetic surfactants form respectively liposomes or surfactant vesicles which are of interest as models for biological membranes,' drug carriers, and drug delivery systems.% Perfluoroalkylated surfactants pose demanding synthesis problems for surfactant chemists. In the course of our studies, we have developed a very flexible method for the synthesis of these compounds which allows the constituent parts, head group, tail, spacer, and connector, to be separately selected from a wide variety of possibilities and varied in a building block or modular fashion. In the literature, few double-chain ammonium amphiphile systems have been reported that have a modular structure. These contain a trifunctional connector derived from glutamic acid,617diethanol amine? or glycerol.9 These amphiphiles all have in common perfluoroalkylchains (provided from perfluoroakyl carboxylic acids or perfluoroalkyl alcohols) grafted on the connector
* To whom correspondence should be addressed.
(1)Fendler, J. H. Membrane Mimetic Chemistry; Wiley and Sons: New York, 1982. (2)Puisieux, F.; Delattre, J. Les Liposomes, Applications Thkrapeutipues; Technique et Documentation Lavoisier: 1985. (3)Ostro, M. J. Liposomes: From Biophysics to Therapeutics;Marcel Dekker: New York, 1987. (4)Gregoriadis, G. Liposomes as Drug Carriers: Recent Trends and Roaress: Wilev and Sons: New York, 1988. 6)Lhger, R. Science 1990,249,1527. (6)Kunitake, T.; Tawaki, S.; Nakashima, N. Bull. Chem. SOC.Jpn. 1983,56,3235. (7)Higashi, N.; Adachi, T.; Niwa, M. J . Chem. SOC.,Chem. Commun. 1988,1573. (8)Kunitake, T.; Higashi, N. Macromol. Chem. 1986,Suppl. 14,81. (9)Santaella, C.; Vierling, P.; Riess, J. G. New J . Chem. 1991,15,685.
through an ester or an ether function. They were obtained in low (20-30%) or moderate yields (50-60%). In this work, we describe a high yielding two-step synthesis procedure for the production of a novel homologous series of surfactants derived from N,N',N'-trisubstituted bis(perfluoroalky1)thiourea (Figure 1). The starting materials were 2-(F-alkyl)-l-iodoethaneswhich have also been recently used for the synthesis of a different set of perfluoroalkylated double-chain amphiphiles.1°-13 A special feature of this modular amphiphile system is the in situ formation of the connector in the synthesis. Furthermore, we report on the surface active and calorimetric properties of the synthesized amphiphiles. The self-assemblybehavior of this new class of perfluorocarbon double-chain surfactants is illustrated by the investigation of the vesiculation abilities under sonication of themember containing CGF13- and C s F l r chains (4h). Experimental Methods 2-(F-alky1)ethylisothiocyanates (1) were prepared from 247alkyl)-1-iodoethanes(a gift from Atochem Company, France) following published procedures.14 3-(Dimethylamino)-N-(2hydroxy-2-(F-alkyl)ethyl)alkylamines (2) were synthesized as described elsewhere.I5Methyl iodide was purchased from Aldrich Chemical Company. Anhydrous acetonitrile and diethyl ether were used as solvents throughout the synthesis procedures. The uncorrected melting pointa were determined on a Bkhi apparatus. Elemental analysis were performed at the CNRS central facilities in Lyon, France. IR spectra were recorded on a Brticker IFS 45 spectrometer. lH NMR and l9FNMR spectra were obtained on a Bruker WH 200 spectrometer at 200 MHz (tetramethylsilane as internal reference) and 84.67 MHz (flu(10)Marty, F.;Rouvier, E.; Cambon, A. (Atochem Co.) French Patent FR 2592648,1987. (11)Marty, F.; Bollens,E.; Rouvier, E.; Cambon, A. J.Fluorine Chem. 1990,48,239. (12)Sismondi, A.; Abenin, P. S.; Cambon, A. (Atochem Co.) French Patent, FR 88 17240,1989. (13)Sismondi, A.; Szbnyi, S.; Abenin, P. S.; Joncheray, L.; Cambon, A. Tens. Surf. Deterg. 1992,29,166. (14)Bollens, E.; Szbnyi, F.; Cambon, A. J. Fluorine Chem. 1991,53, 1. (15)Szbnyi, S.;Vandamme, R.; Cambon, A. J. Fluorine Chem. 1985, 30,37.
0743-7463/93/2409-1201$04.00/00 1993 American Chemical Society
Trabekri et al.
1202 Langmuir, Vol. 9, No. 5, 1993 headgroup
connector HN
R'F
i tails
= Cn Fan+1 R'F = CmFpm+l RF
n
3' 'N'
4, 6
m = 6, 8
RF
R'F
3
p=2,3
Figure 1. Schematicrepresentation of the modular organization of trisubstituted thiourea perfluoroalkylated amphiphiles (RF and R'F indicate perfluoroalkyl chains, p is the number of CH2 groups in the spacer). orotrichloromethane as internal reference), respectively. Mass spectra were run on a Nermag-RibermagR 10-10C spectrometer. Surface Tension Measurements. Surface tension measurements (rs)were performed by an automatic digital tensiometer Prolabo Tensimat N3 employing the Wilhelmy vertical platetechnique. ThesolutioncontainedO.l%(w/w) ofsurfactant in deionized Millipore water and was measured at 25.0 f 0.1 "C. The interfacial tensions (ri)of the amphiphilic solutions were determined toward cyclohexanesolutions by the same technique at 25.0 f 0.1 "C. Differential Scanning Calorimetry. Differential scanning calorimetry was performed on a sample of 10% (w/w) of amphiphile 4h in deionized Millipore water in a Perkin-Elmer 7 series thermal analysissystem. The sample exhibited the milky turbid appearance of lamellar dispersions of double-chain surfactants. The heating rate was 10 "C/min in a temperature range between 10 and 90 "C. The sample was held for 15 min at the end temperatures between repeating runs for equilibration. AH was calculated from the peak areas. Vesicle Preparation and Vesicle Size Determination. To prepare vesicular aggregates, 30 mg of amphiphile 4h were vortexed and equilibrated at 60 "C in 10 mL of deionized Millipore water. After equilibration the sample was sonicated in a bathtype Bandelin Sonorex RK l00H sonicator for both 15 and 30 min at 70 "C, respectively. After cooling, samples were drawn and stained by uranyl acetate (2% (w/v)). The negativestained sampleswere inspected in a Philips EM301 electron microscope. Freeze fracture electron micrographs were obtained in two ways. Samples of sonicated amphiphile 4h were vitrified by propane jet freezing following published procedures.l6 The samples were drawn at 25 OC and at 60 "C. The hot samples were transferred quickly to the freezing apparatus and vitrified. The frozen samples were fractured and shaded by platinum at an angle of 45". The platinum/carbon replicas were inspected in a Philips EM301 electron microscope. Size distribution was determined by extracting the vesicle diameters from the obtained freeze fracture electron micrographs. Quasi-elasticlight scattering was performed on a Malvem 4700 PS/MN spectrometer using a Coherent INOVA 200 argon ion laser (X = 488 nm) as light source. The hydrodynamic radii of the vesicles were determined at 25 OC. The angle dependence of the radii was checked at scattering angles of 45", 60°, and 90". The data were analyzed by second-ordercumulant analysW and exponential sampling inverse Laplace transform program as described by Ostrowsky and co-workers.lsJ9 The samples were centrifuged prior to the measurements to remove dust particles. (16) Miiller, M.; Meister, N.; Moor, H. Mikroskopie ( W e n ) 1980,36, 129. (17) Koppel, D.E.J. Chem. Phys. 1972,57,4814. (18) McWhirster, J. G.;Pike, E. R. J. Phys. A 1978,II,1729. (19) Ostrowski, N.;Sornette, D.; Parker, P.; Pike, E. R. Opt. Acta 1981,28,1059.
CH,l EilO reflux
I
I
RF
R'F
4
Figure 2. Synthetic outline for synthesis of perfluorocarbon compounds 4a-4h. (RF, R'F, p have the same meaning as in Figure 1; r.t. is room temperature).
Synthetic Procedures The overall reaction sequencesfor the synthesisof amphiphiles 4a-4h are depicted in Figure 2. The synthesis of compounds 3h and 4h is described as a general experimental procedure which is applicable to all the compounds in the homologous series.
N-(2-(F-hexyl)ethyl)-N-(2-hydroxy-2-(F-octyl)ethyl)-hr(34dimet hy1amino)propyl)t hiourea (3h). 2-(F-hexy1)ethyl isothiocyanate (lb) (2.83 mmol) in anhydrous CHaCN (10 mL) is added dropwise into a flask containing the compound 2d (2.83 mmol) in anhydrous CH3CN (10 mL). The mixture is stirred for 2 h at room temperature. After evaporation of the solvent and purification over silica gel column with diethyl ether as eluent (R,= 0.136), the residue affords 3h (2.46 g, 90%) as a white powder; F , = 100 OC. N-(2-(F-hexyl)ethyl)-W-(2-hydroxy-2-(F-octyl)ethyl)-N(3-trimethylammonium propyl iodide)thiourea (4h). The compound 3h (4 mmol) in diethyl ether (10 mL) is mixed with methyl iodide (20 mmol). The mixture is heated at reflux of Et20 for 24 h by stirring vigorously. The precipitate is filtered, washed abundantly with EhO, and dried under reduced pressure (1 mmHg at room temperature). Compound 4h is obtained with a yield of 96% (4.26 g) as a white powder; F,,, = 217 "C. Analytical D a t a f o r Compound 3h. Anal. Calcd for C ~ ~ F ~ O H ~ ~C,N29.73; ~ O S F, : 58.79; H, 2.18; N, 4.33; S, 3.30. Found: C, 29.72; F, 58.17; H, 2.01; N, 4.41; S, 3.46. IR (KBr pill) Y (cm-l): 3429 (0-H); 1705 (C=S); 1250-1150 (C-F) . lH NMR (CDC13, 6 [ppml): 9.97, broad singlet, 1 H (NH); 5.80, multiplet, 1H (OH); 4.89, multiplet, 1 H (C6F13-CH,Ht,); 4.51, multiplet, 1 H (CJ?13-CH&,); 4.25, multiplet, 1 H (CHOH); 3.65, multiplet, 2 H (CHzNH); 3.45, multiplet, 2 H (CH(0H)CH2); 2.41, multiplet, 4 H (CH2CH2CH2); 2.25, singlet, 6 H (N(CH3)2);1.82, multiplet, 2 H (CHzCH2CH2). 19FNMR(CDC13,6[ppm]): -81.5 (2CF3);-114.2(CFz,);-122.5 (12F); -123.4 (4F); -124.0 (2F); -126.7 (2CF2,). Mass spectrum (70 eV): m / z 969 (M+, 16.21);58 (CHz=N+(CH3)2, 100); 43 (CH2=NCH3Im+,11.43); 42 (CH2=NtUH2, 12.96);60 ( H z N M + ,9.47);59 (HN=C=Sl'+, 38.84);72 ((CH3)zNCHzCHz+, 25.81); 71 ((CH3)zNCH=CH21'+, 5.36); 70 (CHFN+(CH~)CH=CH~, 10.49); 86 ((CH3)zNCHzCHzCHz+, 15.01);85 ((CH3)2NCH=CH21*+,36.20);84 (CH2=N+(CH3)CH2CH=CHz, 29.86);911 ([CsFi$H(OH)CH21 [ C ~ F I ~ C ~ H ~ N H C S I -
Vesicle Formation of Cationic Surfactants
Langmuir, Vol. 9, No. 5, 1993 1203 Table I. Yields and Melting Points of Compounds 3a-3h compound3 3a 3b 3c 3d 3e 3f 3g 3h a
RF
R'F
p
yield [%I 78 87 75 98 90 71 94 90
Fm"["CI 59 66 79 93 77 80 96 100
Uncorrected values.
Table 11. Yields and Melting Points of ComRounds 4a-4h compound4 RF R'F p yield [%I Fm" ["Cl 96 193 4a 90 190 4b 4c 95 203 210 4d 95 90 204 4e 98 178 4f 91 208 4g 4h 96 217 0
Uncorrected values.
Table 111. Surface Tension T~(water/air) and Interfacial Tension yj (Water/Cyclohexane) of Compounds 4
Figure 3. Freeze fracture electron micrograph of sonicated sample of compound 4h. Bar length is 100 nm. The sample was vitrified by propane jet freezing from 25 " C (below T,= 51.2 "C).
NCH2CH2+,5.24); 910 ([CsF17CH(OH)CH2][C$'13C2H4NHCSlNCH=CH21*+,6.75);69 (CF3+,4.69);936 (M+ - HS, 4.54); 935 (M' - H2S, 4.87). Results and Discussion The presented synthesis procedure stems from our investigations into synthesis strategies for perfluoroalkylated double-chain amphiphiles with ammonium head groupswith or without polymerizablefunctional groups.2o We could show that spherical and unilamellar vesicles could be formed under sonication from these new perfluorocarbon surfactants. In the present report we discuss the synthesis and properties of perfluoroalkylated trisubstituted thiourea amphiphiles. Figure 1gives a schematical representation of the modular organization of these surfactants. The modular organizations allows variations in chain length and degree of perfluorination independently from spacer length and chosen head group. The thiourea connector also has the advantage that it contains a relatively apolar thio function and does not introducean additional polarity close to the tail region. Furthermore the thio group does not contributeto hydrogen bonding. Beside the structural advantages of trisubstituted thiourea, the method has the merits of high yield and a short two-step synthesis. Our choice of perfluoroalkyl chain length was based on the expectation that one CF2 group would equal two CH2 groups in hydrophobicity; therefore the minimum length of C4Fg would be sufficient for bilayer formation. The synthesis strategy was based on the condensation reaction of 2-(F-alkyl)ethyl isothiocyanates (1). Compounds la-lh were prepared followinga versatile synthesis scheme which we introduced re~ent1y.l~ The condensation reaction readily proceeds with 3-(dimethylamino)-N-(2(20) (a) Szcnyi, S.;Watzke, H. J.; Cambon,A. New J. Chem.,in press. (b)Sziinyi, S.; Watzke, H. J.; Cambon, A. Prog. Colloid Polym. Sei. 1992, 89, 149. (c) Sziinyi, S.; Sismondi, A.; Watzke, H. J.; Cambon, A. Jorn. Corn. ESP.Deterg. 1992,23, 219.
comDound4 4a 4b 4c 4d 4e 4f 4g 4h
RF
R'F
p
ys[mN/ml 16.0 16.6 17.0 20.6 18.2 19.2 17.0 20.4
?i[mN/m] 6.4 7.3 9.3 9.2 6.8 9.4 8.9 7.9
hydroxy-2-(F-alkyl)ethyl)alkylamines (2). Compounds 2a-2h were prepared via the oxirane route by coupling of an amine to the 2-(F-alkyl)ethyloxirane. This synthesis strategy was recently elaborated in our 1ab0ratory.l~The second step consists in the quatemization of compound 3 (Table I) by methyl iodide producing compound 4 (Table 11). Both condensation reaction and quaternization proceed with high yields (around 71-98% and 90-98%, respectively). We did not observe the formation of thiocarbamates 3' from the reaction of the 2-hydroxy group of compound 2 with the isothiocyanate 1. We already had made similar observations in reactions using (2-hydroxy-2-(F-alkyl)ethy1)alkylcompounds in other synthesis schemes. The unreactivity of the hydroxyl group seems to be due to its position on the carbon atom bearing the perfluoroalkyl group. Aqueous solutions of 0.1 % (w/w) of amphiphiles 4a-4h exhibit a strong reduction of the surface tension at the air/water interface (Table 111). A significant increase is found at spacer length of p = 3 in combinationwith longer chain length (4dand 4h,Table 111). The interfacialtension between cyclohexane and water is also lowered; the increasing chain length (4f to 4h, Table 111) decreases slightly the interfacial tension. The mutual immiscibility of hydrocarbon and fluorocarbon may be the cause of that decrease. Dispersions of compound 4h in water (10%(w/w))were turbid and of liquid crystalline appearance. Differential scanning calorimetric measurements revealed chain melting (phase transition) temperatures of 51.7 "C (AH = 2.23 kJ/mol) on heating and 27.6 "C (AH = 1.16 kJ/mol) on cooling. No pretransitions were found. The phase transition temperatures are in the expected range for perflu-
1204 Langmuir, Vol. 9, No. 5, 1993
1 0 1 5 20 2 5
Trabelsi et al.
30 3 5 4 0 3 5 5 0 5 5 6 0 6 5 70 7 5 EO 8 5 9 0
Radiuslnm
Figure 4. Size distribution of spherical vesicles obtained from freeze fracture electron micrographs. Samples were vitrified from 25 "C (below T,= 51.2 "C). Table IV. Hydrodynamic Radii of Vesicles Formed by Sonication of 4h at Different Scattering Angle ( t = 25 "C)
scatteringangle [deg] 45" 60" goo
R(h)lnm 32.7 31.3 29.5
polydispersity 0.20 0.16 0.15
orocarbon surfactant bilayers. Despite the shorter chain length and positive head group charge of compound 4h, the Tclies in the range of dipalmitoylphosphatidylserine bilayers (52 oC).21 A strong hysteresis is evident and indicates that strong cooperativity is involved in the chain melting of the bilayer. The self-assembly behavior of compound 4h was tested by studying the vesiculation in dilute solutions by sonication. The low solubility of 4h in water restricted the concentration to 3 mg/mL. The vortexed surfactant solutionstill contained solid particles at room temperature. To start with a evenly dispersed sample, an equilibration at 60 "Cwas necessary. The dispersion was sonicated for 15 min at 62 O C giving a clear and stable solution. Inspection by negative staining and freeze fracture electron microscopy revealed undispersed surfactant particles and vesicular aggregates. The perfluoroalkylatedmaterial can be easily distinguished by the typical decoration effect (specklespattern). Sonication at higher temperature (70 "C) for 30 min resulted in a clear and stable solution. However, freeze fracture electron microscopic inspection of the sample showed the familiar spherical shapes of vesicles only for smaller aggregates; the larger ones were polygon-like shaped (Figure 3). Using the freeze fracture micrographs,we obtained the vesicle size distribution just for spherical aggregates (Figure 4) excluding the polygonlike particles (R = 100 nm). This size distribution has only a qualitative character because of the low number of counted vesicles due to dilution. Quasi-elastic light scattering of the sonicated solutions gave hydrodynamical radii which agreed well with the radius of the size distribution maximum (Table IV). The scattering angle dependence of the determined radii is weak. The moderate polydispersity remains also largely unaffected by the change of scattering angle which shows that the larger aggregateseither exist in the solution only in trace amounts or were produced by the vitrification from 25 "C. Large aggregatesalways strongly influence the polydispersity at lower scattering angle. Therefore the exclusion of the polygon-like and irregular-shaped aggregates appears not to distort the real size distribution. To investigate further (21)Cevc, G.; Marsh, D. Phospholipid Bilayer; Wiley and Sons: New York, 1987.
Figure 5. Freeze fracture electron micrograph of sonicated sample of compound 4h. Bar length is 100 nm. The samples were vitrified by propane jet freezing from 60 "C (above T,= 51.2 "C). Samples contain beside spherical vesicles of radius R = 30 nm also bilayer fragments (A) and large multilamellar aggregates (B).
the occurrence of the polygon-like aggregates, we also vitrified the sonicated samples above the chain melting temperature at 60 OC. If the polygon-like shapes were due to gel state solidification within the bilayers of the larger vesicles, we should observe their disappearance above the chain melting temperature. Figure 5 clearly reveals that this is happening upon heating. However, the size distribution of the smaller vesicles (R= 30 nm)
Vesicle Formation of Cationic Surfactants remains the same as for the sample vitrified from 25 "C (Figure 4 and Figure 5A). Additionally large spherical aggregates appear in the heated solution (Figure 5B) resembling in shape and size multilamellar phospholipid liposomes. The change in shape (spherical to polygonlike or irregular) is well documented in the case of phospholipids. Lepault et and Verkleij and cow o r k e r ~have ~ ~ demonstrated that phospholipids undergo a drastic shape deformation below the T,. Using cry0 electron microscopy they could arrest the different shapes of the liposomes at various temperatures above and below the chain melting transition. The cry0 electron microscopic technique permits the inspection of the vitrified but unperturbed liposome solution. Electron diffraction clearly indicated that in the case of a temperature lower than T, (irregular shaped liposomes), the lipids were in a higher ordered state compared with those above Tc (sphericalliposomes). We can conclude from our findings (Figure 5) that perfluoroalkylated surfactant vesicles behave under heating and cooling similar to phospholipid (22) Lepault, J.; Pattus, F.; Martin,N.Biochim. Biophys. Acta 1986, 820, 315. (23) Frederik, P. M.;Stuart, M.C. A.; B o " , P. H. H.; Busing, W. M.;Burger, K. N. J.; Verkleij, A. J. J. Microsc. 1991, 161, 253.
Langmuir, Vol. 9, No.5, 1993 1205
liposomes due to their high chain melting temperature. Furthermore the results indicate that compound 4h w i l y forms vesiclesunder sonication above ita T,. The constant average size below and above T, adds evidence that the vesicles are preferably forming in sizes of about 30 nm radius. Geometric considerations suggest that these vesicles are unilamellar. The existence of vesicles below and above Tc will permit further studies on the permeability of the perfluorocarbon bilayer depending on the liquid crystalline state of the assembled amphiphiles.
Conclusion The synthesis procedure presented opens a new route to obtaining a large variety of versatile double-chain perfluorocarbon amphiphiles. The modular concept permits the synthesis of surfactanta with different tail length, head groups, spacers, and rigid connectors allowing the investigationof the structural featureson the self-assembly behavior. Studies in this direction are underway in our laboratory. Acknowledgment. The authore gratefully acknowledge the valuable help of Peter Schurtenberger and Ernst Wehrli in light scattering and electron microscopy, respectively.
