Langmuir 1994,10, 3488-3492
3488
Sodium Di-n-dodecyl Phosphate Vesicles in Aqueous Solution: Effects of Ethanol, Propanol, and Tetrahydrofuran on the Gel to Liquid Phase Transition Michael J. Blandamer,*Barbara Briggs, Michael D. Butt, Matthew Waters, and Paul M. Cullis Department of Physical Chemistry, University of Leicester, Leicester LE1 7RH, U.K.
Jan B. F. N. Engberts Department of Organic & Molecular Inorganic Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
Dick Hoekstra Department of Physiological Chemistry, University of Groningen, Bloemsingel 10, 9712 KZ Groningen, The Netherlands
Rajani Kanta Mohanty Department of Chemistry, Visva-Bharati University, Santiniketan 731235, West Bengal, India Received February 22, 1994. I n Final Form: June 23, 1994@ For aqueous solutions containing vesicles formed by sodium di-n-dodecyl phosphate, the gel to liquidcrystal transition occurs near 35 "C, the temperature T,. When ethanol is added, Tmdecreases, but the scan shows evidence of several transitions as more alcohol is added. The effect of added THF and propanol is similar but more dramatic. The scans are similarly complicated if the aqueous solutions are prepared using a solution of DDP in ethanol. However, vesicle ethanol interactions are endothermic although the shift in Tm points to direct binding of alcohol into the vesicle.
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In aqueous solutions, synthetic double-chain amphiphilesl aggregate to form vesicle^.^-^ The amphiphile, di-n-dodecyl phosphate (DDP) as its sodium salt forms closed vesicles6 which are detectable by electron microscopy. The dependence of fluorescence polarization on temperature for DDP(aq) pointed1 to a phase transition a t a melting temperature T , equal to 28 "C. This gelto-liquid crystalline phase transition is an important characteristic property of the bilayer assembly. In the study reported in ref 1,the DDP solutions were prepared by a n ethanol-injection method (see below). However, the protocols for the preparation of DDP vesicles' have a significant impact on the properties of DDP vesicles as revealed by differential scanning microcalorimetry.8 As we demonstrate below by differential scanning microcalorimetry, properties of vesicle solutions prepared using the ethanol-injection method are complex. The scanning microcalorimeter is sufficiently sensitive to identify these complexities associated with either interactions between the cosolvent and vesicles or a change in structure of the aggregates, signaling an advantage over the previously reported fluorescence technique. @AbstractpublishedinAduanceACSAbstracts, August 15,1994. (1)Wagenaar, A.; Rupert, L. A. M.; Engberts, J. B. F. N.; Hoekstra, D. J . Org. Chem. 1989,54,2638. (2) Fendler, J. H. Acc. Chem. Res. 1980,13,7. (3)Kunitake, T. Angezu. Chem., Int. Ed. EngZ. 1992,31,709. (4) Carmona-Ribeiro, A. M. Chem. SOC.Rev. 1992,21,209. ( 5 ) Fonteyn, T. A. A.; Engberts, J. B. F. N.; Hoekstra, D. In Cell and Model Membrane Interactions;Ohki,S., Ed.; Plenum Press: New York, 1991: D 215. _.._ r I
(6)Fonteyn, T.A. A.; Hoekstra, D.; Engberts, J. B. F. N. J . Am. Chem. SOC.1990,112,8870. ( 7 )Carmona-Ribeiro, A. M.; Hix, S. J . Phys. Chem. 1991,95,1812. ( 8 ) Blandamer, M.J.;Briggs, B.; Cullis, P. M.; Engberts, J. B. F. N.; Hoekstra, D. Submitted for publication.
The complex behavior seen by DSC for vesicles prepared by the ethanol-injection method prompted a series of measurements in which controlled amounts of ethanol were added to the vesicle solutions and the DSC scans recorded. Certainly the main extremum shifted to lower temperatures with a n increase in the volume percent of ethanol, but the scans showed additional features. We interpret the patterns in terms of direct incorporation of ethanol into the vesicles. The DSC scans were somewhat more complicated when either THF or propanol was added. In other words, the phenomena seem rather more complex than those reported for the partitioning of apolar solutes into phospholipid bilayer membra ne^.^
Experimental Section Materials. Surfactants and other materials were prepared as described.l
Calorimetry. The differential scanning microcalorimeter (MicroCal Ltd., USA) recorded10 the heat capacities of DDP solutions relative to that of a corresponding solution which contained no DDP. The volume of the cell was 1.2 cm3. A scan rate with rising temperature was approximately 60 K h-l. As previously described,1°a water-water baseline was subtracted from each scan using ORIGIN soflware (MicroCal Ltd.). Therefore, in the figures described below we report the dependence of the differentialisobaric heat capacity dC, (s1n;T)on temperature. A known weight of DDP(s) was added to 2.2 cm3 of water, heated to 55 "C, and held at this temperature for approximately half an hour with stirring. The solution was allowed to cool to room temperature and the required volume of ethanol (L), n-propanol (L), or tetrahydrofuran (L) added. For the aqueous ethanol and aqueous THF systems (where [THF] 5 2 vol %), the (9)De Young, L. R.; Dill, K. A. J. Phys. Chem. 1990,94,801. (10)Blandamer, M. J.;Briggs, B.; Cullis, P. M.; Eaton, G. J.Chem. SOC.,Faraday Trans. 1991,87, 1169.
0743-7463/94/2410-3488$04.50/0 0 1994 American Chemical Society
Sodium DDP Vesicles in Aqueous Solution solutions were placed in the sample cell and cooled to 15 "C. The differential scans were recorded between 15 and 90 "C. For solutions containing 5 2 vol % propanol, the maximum temperature for each scan was 80 "C. For solutions containing 4.8 vol % propanol and 5 vol % THF, the solutions were cooled to 10 "C and scanned from 10to 80 "C. For solutions containing6 vol % and higher propanol, the DDP solutions were cooled t o 5 "C and scanned from 5 to 80 "C. In all cases, the solutions were cooled from the top temperature to the low temperature cited above. The differential heat capacity was then remeasured, in some cases almost immediatelyand, in other cases, after equilibration at the lower temperature for a specified time. The dramatic effect on the differential scans produced by added alcohol prompted an experiment aimed at estimatingthe strength of alcohol-vesicle interaction. Hence, we used a titration calorimeterll in which dilute ethanol(aq) was added in small aliquots to DDP(aq). An Omega Titration Microcalorimeter (MicroCal Inc., USA) was used to record heat q accompanying pulsed injections of aqueous ethanol into a sample cell, volume 1.41 cm3, containing DDP(aq) at 25 "C. The recorded trace comprised a number of extrema showingrates of heating following each injection as a function of time. The data were stored on disk and analyzed later using the Omega software which integrated each peak to yield a plot of heat q as a function of injection number N (see Results section). Preparation of Solutions. With reference to the DSC scans for DDP vesicles in aqueous solutions, we addressed three key issues. First, it was important to ascertain if the trace was reproducible in the thermodynamic sense such that the same pattern was repeated on each heating for a series of heat-coolheat-cool ... sequences. Second, we recorded a trace for several systems after being kept in the calorimeter for 10 or more hours. Third, we compared the traces produced by the calorimeter for DDP solutions having similar concentrations but prepared in different ways. The latter can be classified under three major headings: (I) hot-water method, (11)ethanol-injectionmethod, and for comparisonwith method 11, (111)ethanol-addedmethod. Hot-Water Method. An aqueous solution containing the required amount of DDP was heated to 55 "C. The solution was kept at 55 "C for 1h and cooled t o 15 "C, and the differential scan was recorded by the calorimeter from 15 to 90 "C. A related series of scans were recorded for solutions prepared using Hepes mol dm-3 CH3COONa + Hepes). buffer (5 x (ZZ) Ethanol-Znjection Method. The following procedure was used to prepare, for example, a DDP solution containing 8.42 x mol dm-3. DDP (0.012 g) was dissolved in ethanol (0.12 cm3). A known volume (0.088 cm3)of this solution was injected slowly into 2.2 cm3 of an aqueous Hepes buffer solution (5 x 10-3 mol dm-3 CH3COONa and Hepes), held at 55 "C. The solution was stirred while the DDP solution was added. The DDP solution was held at 55 "C for 1 h. (ZInEthunol-AddedMethod. As we will show below, the scans produced by solutions prepared using methods I and I1 differed considerably. Therefore, comparisonwas drawn with solutions prepared using procedures based on Method I but when ethanol was added prior to recording the scan. DDP(s)was dissolved in the Hepes buffer and kept at 55 "C for 30 min. The solution was cooled to room temperature, and 3.4 vol % of a 95 vol % ethanol water mixture was added. The solution was cooled to 15 "C in the calorimeter. This method was repeated except that 3.4 vol % of ethanol was added to a DDP(aq)in the absence of buffer.
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A most important discovery was the extreme sensitivity of the recorded scans to the method of preparation of DDP solutions. The first series of experiments used method I1 because this method was used in the study reported in ref 1. The complexities of the DSC scans and particularly the lack of reproducibility prompted a detailed investigation of the dependence of observed gel to liquid crystalline transition on the method of preparation of the vesicle (11)Blandamer, M. J.;Briggs, B.; Butt, M. D.; Cullis, P. M.; Waters, M.;Engberts, J. B. F. N.; Hoekstra, D. J.Chem. SOC., Faraday Trans., in press.
Langmuir, Vol. 10, No. 10, 1994 3489
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Figure 1. Dependence on temperature of the differential heat (monomer mol) dm-31 capacity 6C, for DDP [aq; 8.4 x buffered solutions prepared using method I. Plots a-d were recorded after the solutions were cooled to 15 "C and scanned to 90 "C. Plot e was recorded 10 h after scan d. solution. We illustrate the point by concentrating attention here on DDP solutions having the same monomer (monomer mol) dm-3. concentration, 8.4 x The scans in Figure 1 were recorded for buffered solutions prepared using method I in which, following preparation, the solution was cooled in the calorimeter to 15 "C and the differential heat capacity scanned from 15 to 90 "C. Each solution was then cooled to 15 "C and then re-scanned. The scans were fully reversible. A fifth scan recorded 10 h later showed a n extremum at 44 "C, but in all cases, the dominant feature was the extremum at 35 "C. The DSC scans for independently prepared samples using method I were fully reproducible. Similar reproducibility was again recorded for aqueous solutions prepared in the absence of buffer. In other words, the features associated with the gel to liquid crystal transition were not sensitive to the presence ofbuffer. Consequently, solutions prepared using method I (but containing no buffer) established a foundation against which to compare the results of other experiments and methods of preparation. The scans recorded using method I1 were significantly different from those shown in Figure 1. An extremum was recorded near 35 "C, but it was much less marked. I n addition, a broad extremum was recorded near 31 "C (Figure 2). In these experiments the solutions after preparation were placed in the calorimeter and cooled to 15 "C, and the differential heat capacity was recorded for the range 15-90 "C. I n each set of experiments the first scans differed from subsequent scans on the same solution. The first scan for each freshly prepared solution was also not reproducible between independently prepared samples. Scans recorded for DDP solutions prepared using method I11 were again different (Figure 3). The original extremum near 35 "C lost most of its intensity whereas an extremum at 31 "C (cf. method 11)was the dominant feature. The scans were reproducible upon re-scanning (Figure 3) over five consecutive scans. A scan after 11h showed evidence for an underlying change. The purpose of the experiments described above which involved addition of ethanol to the aqueous DDP solutions was to contrast the resultant scans with those where ethanol was used directly in the preparation of the solutions. These results prompted a more detailed examination of the effect of added ethanol on the DSC
Blandamer et al.
3490 Langmuir, Vol. 10, No. 10, 1994
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Figure 2. Dependence on temperature of the differentialheat (monomer mol) dm-31: (a)DDP capacityofDDP [sln;8.4 x solution prepared by ethanol-injection method (method 11) where the mole fraction of ethanol is approximately 0.01 (3.9 ~ 0 1 % )(b) ; DDP solution prepared using method I (see Figure 1);(c) DDP solution prepared using method IIIa with an extremum at 31 "C.
Figure 4. Differential heat capacities of DDP (aq; 8.4 x lov3 mol dm-3)containing2 vol % ethanol. The scans were recorded (a) after the samples were prepared and cooled in the calorimeter, the extrema being at 32,36, and 45 "C,(b-e) after they were cooled from 90 to 15 "C, and (f,g) aRer they were cooled and equilibrated at 15 "C for 11h. The plots have been displaced for clarity on the heat capacity axis.
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Figure 3. Dependence on temperature of the differential he,. mol dm-3) prepared (method capacity of DDP (sln; 8.4 x 111)by adding 3.9 vol % ethanol to an aqueous solution of DDP. Scans a-e were recorded consecutively, and scan fwas recorded 11h after scan e. scans. Almost without exception, the first recorded trace for each freshly prepared solution differed from the next set of recorded scans. In the example shown in Figure 4, the first scan for a DDP solution containing 2 vol % ethanol shows extrema a t 32,37, and 46 "C. The following four recorded scans are dominated by the main feature a t 32 "C. This feature was reproducible using freshly prepared solutions, and so we concentrate our attention on these extreme in the recorded scans. In some cases, we allowed the DDP solutions to remain in the calorimeter for periods of 11 h or more. The recorded scans often showed new features (cf. Figure 4), but these were not reversible, and so we do not comment on them further. In general terms, the temperature extremum a t 35 "C for DDP(aq) moved to lower temperatures with an increase in the volume percent of ethanol over the range 0 Ivol % 9 9.0 (Figure 5). The scans recorded for solutions containing higher concentrations were extremely complicated and not reproducible. In all cases, the concentration of alcohol in these DDP solutions was greater than the concentration of DDP. With an increase in the volume percent of ethanol a t and above 7.4, a new feature
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Figure 5. Differential heat capacities of DDP (aq; 8.4 x mol dm-3) containing increasing volume percents of ethanol which had been added subsequent t o solution preparation: (a) 0 vol % with extremum at 35 "C, (b) 2 vol % with extremum at 32 "C, and (c) 3.9 vol % with extremum at 31 "C and (d) 7.4 vol % and (e)9 vol % both with extrema at 24 and 32 "C. The plots have been displaced for clarity on the heat capacity axis. developed with a n extremum near 32 "C. So, for example, a solution containing 9 vol % showed two extrema a t 28 and 32 "C. The extent to which the patterns in Figure 5 are a consequence of direct ethanol-vesicle interaction was studied using titration calorimetry. Here, small aliquots of ethanol(aq) were added to DDP(aq). A typical titration plot is shown in Figure 6a, which comprises a sequence of exothermic peaks, each pair of peaks being separated by a small baseline. We conclude that there is no slow kinetic process and that the sequence of peaks characterizes a series of equilibrium states. However, a blank experiment was completed in which comparable exothermic pulses were recorded when an identical aqueous ethanol solution was injected into a reservoir containing no DDP (Figure 6b). A slight difference was just discernible from the integrated plots in which the water-only plot was subtracted from the pattern produced by the DDP system (Figure 6c). The latter shows that the ethanol-
Sodium DDP Vesicles in Aqueous Solution
Langmuir, VoE. 10,No. 10,1994 3491
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Figure 7. Differential heat capacities of DDP (aq; 8.4 x mol dm-3)containing2vol % propanol; five repeat scans showing extrema at 30 and 33 "C. 0.08
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Figure 8. Differential heat capacities of DDP (aq; 8.4 x mol dm-3) in (a) aqueous solution, (b) aqueous ethanol (2 vol %) where T, = 32.2 "C, (c)'aqueousTHF (2 ~ 0 1 % where ) T, = 31 "C for the main extremum, and (d) aqueous propanol (2 vol %) where T, = 30 "C for the main extremum. The plots have been displaced for clarity on the heat capacity axis.
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Figure 6. Titration calorimetry at 25 "C: (a, top) exothermic pulses accompanying injection of aliquots of 5 vol % ethanolmol dm-3);(b, middle) exothermic (as) into DDP (aq;8.4 x pulses accompanying injection of aliquots of 5 vol % ethanol(aq)into initiallywater in the sample cell; (c, bottom) difference in integrated plots obtained from the injection pattern parts in a and b. DPP interaction is slightly and perhaps surprisingly endothermic. When propanol was added to DDP(aq), the DSC traces a t low volume percents of alcohol were similar to those described above (Figure 7). With an increase in the volume percent of propanol, the extremum shifted to lower temperatures, the shift being the more dramatic than for those systems containing ethanol. However, when the volume percent of propanol was greater than or equal to
4.8, the traces were extremely complex. As shown in Figure 7, the traces are reproducible for solutions containing 2 vol % propanol, having two extrema a t 30 and 33 "C. The main feature shifts gradually to lower temperature with a n increase in the volume percent of propanol, although there are many other small extrema a t higher temperatures. When THF (2 ~ 0 1 %was ) added, a similar pattern emerged with two extrema a t 31 and 34 "C, intermediate between those recorded for the DDP solutions in aqueous ethanol and aqueous propanol (Figure 8). With a further increase in the volume percent of THF, a complicated scan was recorded similar to that obtained when the volume percent of propanol was increased.
Discussion Many previous studies have confirmed that di-n-alkyl phosphates readily form unilamellar vesicles.l,' In the DSC scans for the DDP(aq) vesicles, the extremum near 35 "C is assigned to a gel to liquid crystal transition. In other words, the n-dodecyl chains within the bilayer "melt" and gain local With respect to the properties of surfactants there is considerable interest in how the structure of the monomer and the solution composition determine the nature of the aggregates. In this study we
3492 Langmuir, Vol. 10,No. 10,1994 have demonstrated that DSC can detect the effects of added cosolvents on vesicle structure a t relatively low concentrations ( < 5~ 0 1 % ) .In principle, the effect of added cosolvents could be either direct, by incorporation into the bilayer, or indirect, by influencing the size or nature (multilamellar closed vesicles, open planar lamellar structures, etc.) of the aggregate. It has previously been shown that the size of vesicles can vary, according to the method of preparation.12J3 Aqueous solutions of DDP vesicles prepared by method I are well behaved in terms of DSC, and studies by electron microscopy are consistent with rather uniform closed vesicles being present.8 Vesicle solutions prepared by method I1 show DSC properties closely similar to those for solutions prepared by method I11but only after several thermal cycles. Addition of ethanol to a vesicle solution prepared by method I, i.e. the protocol used for method 111, leads to a n immediate shift of the major transition from 35 to 31 "C. No further changes take place through repeated thermal cycles. This result suggests a direct incorporation of ethanol into the vesicles and, furthermore, implies that this ethanol penetration can occur readily with vesicles in the gel state. In view of these observations, the complexities seen in the initial DSC scans of vesicles prepared by method I1 are unlikely to be due solely to the direct interaction of ethanol with the vesicle but, presumably, signal changes to the nature of the aggregate formed initially. Thermal cycling brings this to the same "equilibrium" state as obtained by method 111. It is interesting to note t h a t all vesicle solutions containing ethanol when left a t room temperature for many hours ('12 h) show additional complexities which are again lost on repeated thermal cycling. This "aging" of the solutions suggests t h a t there is a very slow reversible reorganization of the vesicles in solutions a t room temperature induced by ethanol and that the equilibrium detected after repeated thermal scans may not be a true equilibrium state a t the low-temperature end. The shiR in T,,, from 35 to 31 "C in the presence ofethanol appears most likely to be associated with direct interaction of ethanol with the vesicle. The extent of this binding of ethanol within the bilayers determines the vesicle prop(12) Lasic, N.N.Biochem. J. 1988, 256,1. (13) Cuccovia, M.;Feitosa, E.;Chaimovich,H.; Sepulveda, L.; Reed, W. J.Phys. Chem. 1990,94,3722.
Blandamer et al. erties. The results show that it is difficult to reproduce the initial aggregate state from sample to sample when it is prepared by ethanol injection since this presumably will be influenced by the efficiency of mixing and the kinetics ofthe aggregation process. In fact, the extremum at 31 "C, more apparent after thermal cycling, shows that, in the bilayer domains which contain ethanol molecules, the gel to liquid transition is significantly perturbed. In the series of experiments involving DDP in aqueous ethanol, the gradual change in the pattern of the dependence of SC, on temperature through a series of repeat scans is consistent with a facile incorporation of alcohol in the liquid crystal form ofthe vesicle. Nevertheless, the first scan showed that DDP vesicles had taken up ethanol even when the DDP vesicles were in the gel phase. The observed changes in T , are consistent with a model in which the vesicles comprise patchwork domains of grouped monomers. This model accounts for the changes in the scans when either THF or propanol is added. Interestingly, these more hydrophobic solutes (relative to ethanol) are incorporated to a larger extent within the vesicle bilayer. The two extrema in plots of SC, against temperature would be consistent with there being domains within the vesicle bilayer which are alcohol-rich and domains which contain relatively small amounts of added solute. The shift of T , to lower temperatures with an increase in the volume percent of alcohol signals a decrease in the thermal stability of vesicles in the gel phase. As commented above, the transition a t 35 "C for DDP vesicles is attributed to a gel to liquid crystal transformation. The titration calorimetry has also shown direct interaction between ethanol and vesicles even in the gel state. The endothermic pattern formed by integrated plot (Figure 6c) was surprising. However, extensive studied1 involving titration calorimetry using dimethyldi-n-octadecylammonium bromide vesicles in aqueous solution shows that this endothermic trend is not unexpected.
Acknowledgment. We thank the University of Leicester for a travel grant to M.J.B. and the SERC for their support under the Molecular Recognition Initiative; we thank the Royal Society for a grant given to P.M.C. for the purchase of the Titration Microcalorimeter; R.K.M. thanks the Department of Chemistry (Visva-Bharati University) for a leave of absence.
