5892
J . Phys. Chem. 1986, 90, 5892-5895
Hydrocarbon Gels from Water-in-Oil Microemulsions Gabriel Haering and Pier Luigi Luisi* Institut fur Polymere, Eidgenossische Technische Hochschule, 8092 Zurich, Switzerland (Received: April 7, 1986)
A procedure is described that permits the transformation of hydrocarbon microemulsions into gels. This is obtained by cooling a previously heated isooctane solution of bis(2-ethylhexyl) sodium sulfosuccinate containing 10-20% of an aqueous gelatin solution. The final system is a homogeneous gel, whose consistency and physical properties vary depending upon the relative concentration of water and gelatin. These new hydrocarbon gels have been investigated with 'H NMR, circular dichroism, differential scanning calorimetry, and electron microscopy. Enzymes and bacteria can be entrapped in apolar gels without loss of activity.
Introduction Several papers have appeared describing the properties of proteins hosted in reverse micelles.'-5 The system of choice is bis(Zethylhexy1) sodium sulfosuccinate (AOT)/isooctane/water, due mostly to the fact that this system permits water solubilization over a wide range of w, (w, = [HzO]/[AOT]) values6g7(at high w, values the term water-in-oil microemulsions is more accurate than reverse micelles). As a result of the micellar environment, proteins acquire novel physical and conformational properties, which has lead to an interesting research perspective from both the biophysical and the biotechnological points of In this paper, we will describe a particular effect caused by the solubilization of gelatin in water-in-oil microemulsions. As is well-known, gelatin is able to form gels in water.15 This is generally obtained by cooling concentrated solutions from above 40 "C to approximately 35 "C or below, a phenomenon that is the basis of an industrially important process and one that is very interesting from the point of view of structural protein chemistry. Since gelatin can be solubilized in reverse micelles,I0 it appeared interesting to investigate how the micellar environment affects this protein gelling behavior. Our expectation was that gel formation would take place within the water pools of the reverse micelles, eventually bringing about a clouding of the hydrocarbon micellar solution and possibly phase separation of the aqueous gel. Something completely different takes place: the whole micellar solution becomes a gel. This paper describes the preparation of these novel materials and their basic physical properties and gives indications of some possible application in the area of applied biochemistry. Experimental Section Isooctane (puriss. p.a.), gelatin (Bloom 250), and cytochrome ( I ) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Pantin, V. I.; Berezin, I. V. Biochim. Biophys. Acta 1981, 657, 277. (2) Douzou, P.; Keh, E.; Balny, C. Proc. Natl. Acad. Sci. U.S.A.1979, 76, 681. (3) Fletcher, P. D. I.; Freedman, R. B.; Mead, J.; Oldfield, C.; Robinson, B. H. Colloid Surf. 1984, 10, 193. (4) Levashov, A. V.; Klyachko, N. L.; Pantin, V. I.; Khmelnitsky. Y. L.; Martinek, K. Bioorg. Khim 1980, 6, 929. (5) Zampieri, G.; Jickle, H.; Luisi, P. L. J . Phys. Chem. 1986, 90, 1849. (6) Menger, F. M.; Donohue, J. A.; Williams, R. F. J . Am. Chem. SOC. 1973, 95, 286. (7) Eicke, H. F.; Shepherd, T. C. W.; Steinemann, A. J . Colloid Interface Sei. 1976, 56, 168. (8) Barbaric, S . ; Luisi, P. L. J. Am. Chem. Soc. 1981, 103, 4239. (9) Luisi, P. L. Angew. Chem. 1985, 97, 449-460. (IO) Luisi, P. L.; Imre, V. E.; Jackle, H.; Pande, A. in D. D. Breimer, P. P. Speiser: Top. in Pharm. Sei., 1983, 243. (11) Hilhorst, R.; Laane, C.; Veeger, C. FEES Lett. 1983, 159, 31. (12) Luisi, P. L.; Laane, C. Trends Eiotechnol. 1986, 4(6), 153. (13) Birrenbach, G.; Speiser, P. P. J. Pharm. Sei. 1976, 65(12), 1763. (14) Speiser, P. P. Applied Biology and Therapeutics; Lingle, I. T.; Schoch, P. P.; Shore, I. W.; Eds.; Elsevier: Amsterdam, 1979; Vol. 6. ( 1 5) Veis, A. The Macromolecular Chemistry of Gelatin; Academic: New York. 1964.
0022-3654/86/2090-5892$01 S O / O
c were from Fluka (Switzerland) and were used without additional purification. AOT was from Serva (ControlE). 5-Bromo-4chloro-3-indolyl galactopyranoside (x-gal) and isopropyl 0-Dthiogalactoside (IPTG) were from Bohringer, Mannheim (Germany). Strain Escherichia coli K12-7 1/ 18 harboring plasmid PEMBL 8, constructed and described by Dente et a1.,I6 was a gift from Dr. D. Clark, Washington, DC. Preparation of the Gels. Gelatin in the solid state is added to the micellar H,O/AOT/isooctane solution at 40-50 "C under vigorous stirring. First, this hot mixture is cooled at ca. 30 "C under mixing and stirring till it becomes very viscous and homogeneous. Then, this cloudy viscous mixture is left standing at room temperature till it becomes a clear gel. Gelatin is expressed in water pool (wp) and overall (ov) percentage in grams per 100 mL. IH NMR spectroscopy was performed with a Bruker HXS 360 in the presence of an external standard of Me4Si/acetone. Differential-scanning calorimetry was measured with a Perkin-Elmer (DSC-2B) cooled with liquid nitrogen and then recorded with a scan rate of 5 "C/min from -50 to +70 "C. Viscosity measurements were carried out with a Rotovisco Haake RV2 at room temperature. Circular dichroism (CD) was measured with a Jasco 5-40 AS. The measurements were carried out with ge! films (0.1 mm f 0.002 mm) contained between two glass plates and in a cell of 0.1 mm for the liquid gelatin microemulsion. Transmission Electron Microscopy ( T E M ) . The samples (20-pm films) were frozen in a propane-jet freezer between 0.1-mm copper plates with a cooling rate of 10000-20000 K s-I.I7 Freeze-fracturing was performed in a Balzer BAF 300 apparatus at -155 "C and mbar. Replicas of the fracture faces were obtained by shadowing of 2-nm Pt/C from an angle of 45" and subsequently of 20-nm C from an angle of 90". The replicas were cleaned for 30 min in 70% HzS04and then observed in transmission electron microscopy (Philips 30 1). Scanning Electron Microscopy (SEM). The samples were frozen and fractured in the same way as described above. They were then freeze-dried for 2 h at -80 "C and mbar. After the specimens were warmed to room temperature, they were rotary shadowed with 4-nm Pt/C at an angle of 45" and observed in a Hitachi S700 scanning electron microscope equipped with a field emission electron gun.
Results The procedure for preparing hydrocarbon gels from AOT hydrocarbon solutions is analogous to that used for preparing an aqueous gel from gelatin: gelatin is added to the hydrocarbon micellar solution at around 40-45 OC, and after the mixture cools under stirring, the gel forms. The gelation of the microemulsion (16) Dente, L. D.; Cesarini, G.; Cortese, R. Nucleic Acids Res. 1983. 1I , 1645-1 655. (17) Muller, M.; Meister, N.; Moor, H. Mikroskopie 1980, 36, 129-140.
0 1986 American Chemical Society
The Journal of Physical Chemistry, Vol. 90, No. 22, 1986 5893
Hydrocarbon Gels from Water-in-Oil Microemulsions
I
IV
IV
2
I
X
a 30'
'
1
'
20
40
A
20
40
E
W O
Figure 1. Stability regions of the AOT/isooctane gels, with (A) 100 mM and (B) 140 mM AOT. The four regions represents the different states of the micellar solutions: (I) micellar gelatin liquid solution; (11) gel phase; (111) phase separation (the apolar solvents separates out of the gel); (IV) phase separation of solid gelatin.
TABLE I: DSC of Micellar AOT/Isooctane/Water/Gelatin Gels [AOTI w, Q G,, %, G T," A€P 50.7 34.3 31.5 100 40 7 48 37.1 26 150 40 10 50.5 37.3 32 150 40 11 56.5 150 30 10.5 38.3 26.6 30 11 57.6 38.3 25 150
P Y m I
z X
L
a I n
B,
Figure 2. Circular dichroism spectra as a function of temperature and aging of a gel of 4.4% G,, w, = 60 and [AOT] = 100 mM: (1) dena-
tured gelatin at 40 OC; (2) after the cell was rapidly cooled to room temperature (ca 10 min) and measured after 1 h; (3) after 19 h; (4)after 42 h; (5)after 5 days.
"The onset temperature was chosen as the melting temperature T,. *The value is given in joules per gram of polymer. does not take place however at all concentrations of gelatin and/or water. At low w, values, formation of gels is not possible, probably because not enough gelatin can be dissolved in the system. Actually, we could not obtain a gel at w, lower than 20 regardless of the surfactant concentration. Figure 1 shows the relationship between water and gelatin concentration that permits the formation of gels a t different w,, gelatin content, and AOT concentration. Generally, the protein must be slightly in excess of its maximal solubility in the micellar system-in other words the micellar system with added gelatin is generally slightly cloudy. It must be noted that also the concentration of AOT should be above a certain critical value; for example, under our conditions we could not obtain gels at 50 mM AOT. The lowest gelling region for an AOT concentration of 100 mM is observed with 4.5% water and 6.5% gelatin (overall concentrations). The phenomenon of gel formation is reversible: by heating the gels above 40 O C , they melt, and this solution can be transformed again into a gel, provided that care is taken to stir during the cooling-otherwise there is phase separation. Gels appear to be indefinitely stable upon standing in closed vessels. In open air, due to water and/or hydrocarbon evaporation, there is a change of the composition, and gels tend to become stiffer. Concerning the stability, it is however interesting to notice that the gels obtained from isooctane are insoluble and actually quite stable also mechanically in isooctane or similar hydrocarbon solvents. Despite the initial cloudness of the microemulsions at 40 O C , gels in their final form are generally transparent. This permits electronic spectroscopy to be performed: for example, the peptide chromophoric region of the protein can be monitored down to 210 nm. Figure 2 shows CD spectra of a gel after heating at 40 OC (curve 1) and shows that the refolding process is complete after a few days. It is apparent that the helical content increases with aging, as is the case for aqueous gelatin gels. The physical appearance of the gels and their physical properties depend on the composition. In Table I a series of different gels are compared. In particular, we have measured transition temperatures with differential scanning calorimetry (DSC). It is possible to observe that the heat of transition is not very sensitive to the composition of the system. The transition temperature T,,,is around 34-38 "C and is, like
6(ppm) 4.8
4.7
4.6
Figure 3. 'H NMR of water in a micellar system of isooctane/AOT/ water and gelatin (AOT = 150 mM): (A) liquid microemulsion with 4.5% Go";(B) gel with 9% Go".
AH, close to that reported for gelatin in at the same overall concentration. An interesting question is whether and to what extent water molecules in the gels have restricted flexibility. NMR is the most obvious technique to investigate this problem, and Figure 3 shows the proton resonance of water in the gel compared to that of the starting microemulsion solution. The much broader signal in the case of the gel is a clear indication of the more restricted mobility of water in the gel phase. Generally, the mobility of water decreases by increasing the content of gelatin, all other parameters being the same. A quantification of this effect as a function of ~
(18) Borchard, W.; Brenner, W.; Keese, A. Colloid Polym. Sci. 1980, 258, 516. (19) Tomka, I. Chimia 1983, 37(2), 33. (20) Hayashi, A.; Chol-Oh, S . Agn'c. Biol. Chem. 1983,47(8), 1711-1716.
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Haering and Luisi
Figure 4. Micrographs of freeze-fractured samples taken by the TEM (A, B; sign = 1000 A) and micrographs of freeze-dried samples taken by SEM (C, D; sign = 2000 A) of gelatin in the system AOT ( 1 50 mM), isooctane, and water (w,,= 40): (A, C) gel with 9% G,; (B, D) liquid micellar solution with 4.5% G , .
the gel composition and other parameters (e.g., temperature) is in progress. The study of the structure of the gels by electron microscopy (EM) proved to be difficult, as the classical techniques in use are not able to cope with hydrocarbon matrices. TEM pictures of both the freeze-fractured microemulsion solutions (Figure 4A) and the freeze-fractured gels (Figure 4B) indicate that in the gel phase we have a network of tubular-like structures (with a diameter of about 200 A). These tubules might represent gelatin fibers connecting various water pools with each other. That is the opposite of the micellar solution were we have spherical globules. These spherical particles show a diameter of about 150-300 A that confirms the values founded by light scattering:' small-angle X-ray diffraction,22and pulse radiolysis23 for micellar solutions of AOT and water in isooctane without gelatin. SEM pictures of the freeze-dried samples confirm this finding. They show a three-dimensional network of tubules in the gel phase (Figure 4C) and globular structures in the micellar phase (Figure 4D) although, in the latter case, a collapsed structure due to the freeze-drying process was expected. Enzymes and other biopolymers can be added together with gelatin before the gelatin process is terminated and they remain homogeneously entrapped in the gels. This can be checked spectroscopically or simply by visual inspection in the case of proteins that absorb in the visible, such as cytochrome c or myoglobin. (21) Zulauf, M.; Heicke, H. F. J. Phys. Chem. 1979, 83(4), 480-486. (22) Pileni, M. P.; Zemb, T.; Petit, C. Chem. Phys. Lett. 1985, 118(4), 414-420. (23) Pileni, M. P.; Brochette, P.; Hickel, B.; Lerebours, B. J . Colloid Interface Sci. 1984, 98, 549.
Figure 5 shows the spectral investigation and circular dichroism spectrum of the Soret band of cytochrome c immobilized in the hydrocarbon AOT gels. The spectrum in the gel phase is quite similar to that in micellar solution and is comparable with the different spectra found by Takeda24with sodium dodecyl sulphate (SDS) and water solution, showing similar interactions of AOT and SDS on cytochrome c and the basic structural integrity of the enzyme in the gel film. Not only enzymes but also whole bacterial cells can be entrapped in the apolar gels. It has been reported before that bacterial cells can be solubilized in organic solvents with the help of reverse micelles.25 When gelatin is added to similar microemulsion solutions and the gelation process is induced as described above, a homogeneous dispersion on @-galactosidaseinduced E. coli (with IPTG) in the gel can be obtained. The gels remain transparent, and it has been possible to show that the bacteria maintain the enzymatic activity for an even longer period, as indicated by the hydrolysis of x-gal.
Discussion The gelation of a microemulsion consisting, typically, of more than 80% hydrocarbon solvent is quite a surprising phenomenon. The mechanism of the gelation and the structure ensuing from this phase change are still rather obscure, and only some reasonable guess can be made at the present. One can for example assume that gelatin, a material that is soluble in water and insoluble in the organic phase, is initially confined overwhelmingly in the water (24) Takeda, K.; Takahashi, K.; Batra, P. P. Arch. Biochem. Biophys. 1985, 236,411-417.
( 2 5 ) Haering, G.; Luisi, P. L.; Meussdoerffer, F. Biochem. Biophys. Res. Commun. 1985, 127(3), 91 1-915.
Hydrocarbon Gels from Water-in-Oil Microemulsions
-301 I
.
360
380
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420
A
440 nm
Figure 5. Circular dichroic spectra of cytochrome c in water (--), in micelles (-) with w, = 50 and in gel (---) with w, = 50 and 7% G,.
pools of the reverse micelles. It should borne in mind however that a t the relatively high concentration of AOT used in our experiments (100 mM or higher) the distance among the micelles in the microemulsion solution is small, of the order of the diameter of the micelles, Le., ca. 100 A at w, = 30. Is then reasonable to assume that extensive intermicellar contacts and percolation26 are present; since the gelatin is slightly in excess of its solubility in the water pools but mostly confined to the water microphase, one can envisage the system as one in which the long gelatin molecules are bridging the micelle water pools with each other. Initially, this takes place in a dynamic way, (26) Cazabat, A. M.; Chatenay, D.; Guering, p.; Langevin, D.; Meunier, J.; Sorba, 0. Surfactanrs Solution 1982, 3, 1737.
The Journal of Physical Chemistry, Vol. 90, No. 22, 1986 5895 with a rapid equilibrium among very many possible configurations; then, as a result of the gelation of gelatin in the water microphase, the whole continuous system is rigidified as a homogeneous continuous network. This qualitative picture, based on micellar percolation and gelatin bridging, does not quite explain how the large hydrocarbon mass is able to gel. In particular, the thermodynamic description of the transition appears very complex in view of the fact that extensive stirring is necessary for the formation of the gels-which indicates that kinetic effects also play an important role. These gels are novel materials, with quite surprising properties. For example, they are insoluble in the very hydrocarbon solvent from which they originate (isooctane). In the literature, mostly in the patent bibliography, there are several examples of polymer-induced gels in organic solvent^.^^*^* These are generally obtained by a very large concentration of polymer. In our case, the overall gelatin concentration can be as low as 5%, and most importantly, these gels originated from water-in-oil microemulsions behave 4s microheterogeneous systems. In other words, they can contain water-soluble substances (suck as enzymes, but of course also low molecular weight salts or other hydrophilic molecules) homogeneously dispersed in the bulk hydrocarbon phase-precisely like reverse micellar solutions do. This compartmentalization perinits interesting chemical applications, as we have seen in the case of bacteria. Also the spectroscopic investigation of protein in a rigid matrix seems to be quite feasible with these systems. The entrapment of enzymes in apolar gels may lend itself to an interesting biotechnological application for the bioconversion of those water-insoluble substrates that are soluble in hydrocarbon or in other organic solvents. Some specific applications are mentioned in recent patent.29
Acknowledgment. We acknowledge with much appreciation the skillful assistance of E. Blijchlinger, and Dr. W, Genshuan, the collaboration of Dr. E. Wehrli (Institut fur Zellbiologie) for electron microscopy, of Dr. H. Jackle and F. Bangerter for NMR, and of Dr. I. Tomka for interest and advice. Registry No. AOT, 577-1 1-7; isooctane, 540-84-1.
(27) Gay, L. R.; Schlott, J. R.; Burroghs, E. J. USA Patent, Chem. Abstr. 90-074182/10. (28) Akira, I.; Mitsuru, T.; Kikuo, T.; Yasumasa, H. Japanese Patent, Chem Abstr. 86-092993114. (29) Luisi, P. L. Swiss Patent PCT/CH85/00154, with priority date Oct 1984.
