J. CHEM. SOC. FARADAY TRANS., 1994, 90(13), 1905-1907 I905 Differential Scanning Microcalorimetric Study of Sodium Di-n-dodecylphosphate Vesicles in Aqueous Solution Michael J. Blandamer, Barbara Briggs and Paul M. Cullis Department of Physical Chemistry, University of Leicester, Leicester, UK LEI 7RH Jan B. F. N. Engberts Department of Organic and 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 The scans recorded by differential scanning calorimetry are reported for aqueous solutions containing vesicles formed by sodium di-n-dodecylphosphate (DDP). The gel-to-liquid-crystal transition occurs near 35 "C,the melting temperature T,.The dependences of heat capacity on temperature near T, are analysed to yield related enthalpies of transition and patch numbers which record the number of DDP monomers which melt co-operatively. The dependences of the enthalpy of transition and patch number offer indications of vesicle size and the tightness of packing of monomers within the vesicles. In aqueous solutions, synthetic double-chain amphiphiles' aggregate to form These are interesting systems because they resemble natural membrane system^.^ The amphiphile, di-n-dodecylphosphate (DDP) as its sodium salt forms vesicles as confirmed by electron microscopy.6 Each vesicle of DDP has a diameter' less than 100 nm although the related C,,/C,, derivative can form vesicles having sig- nificantly larger diameters; e.g.1000 nm. The related di-n- hexadecylphosphate (sodium salt) can form7 vesicles using a chloroform injection method; these vesicles have a mean diameter of 270 nm. The dependence of fluorescence polariza- tion on temperature for DDP(aq) indicated' a phase trans- ition at a melting temperature T, equal to 28 "C. In the study reported here we used differential scanning microcalorimetry to study the gel-to-liquid-crystal transition for DDP(aq) in dilute solutions. The technique is sufficiently sensitive to allow the properties of DDP to be measured in aqueous solu- tions for which one can be sure that individual vesicles are comparatively far apart.Consequently, the measured param- eters are likely to describe intra-vesicular properties with only minor contribution from inter-vesicular interactions. These conclusions follow from simple calculations described pre- viously,' based on a model for solutions described by Robin- son and stoke^.^ In reviewing the patterns which emerge from these DSC studies, we use the previously discussed7 model for the vesicle structure that involves patches of monomers, the vesicle sur- faces comprising aggregates of these patches. In these terms, the gel-to-liquid-crystal transition for DDP in aqueous solu- tion is a co-operative processlo." involving over one hundred DDP molecules where [DDP] z 8.4 x mol dm-3. This transition is accompanied by a change in organ- ization of the charged head group.Experimental Materials Surfactants and other materials were prepared as described. ' Calorimetry The differential scanning microcalorimeter (MicroCal Ltd., USA) recorded12 the heat capacities of DDP solutions rela- tive to that of a corresponding solution which contained no DDP. The volume of the cell was 1.2 cm3. Temperature was increased at ca. 60 K h-'. As previously described,I2 a water-water baseline was subtracted from each scan using ORIGIN software (MicroCal Ltd.). Therefore, in the figures described below we report the dependence of the differential isobaric heat capacity SC, (sln; 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 cu.30 min with stirring. The solution was allowed to cool to room tem- perature placed in the sample cell of the calorimeter. (For further details see the Results section.) Analysis of Heat Capacity Data In the event that an extremum is observed in the dependence of SC, on temperature, the simplest model attributes the 'bell-shaped' plot to a two-state chemical equilibrium; X (aq)=Y(aq) characterised by equilibrium contant K at temperature T and a standard enthalpy of reaction, ArHe. Hence the dependence of the molar heat capacity C,, on temperature is given by eqn. (1). C,,(T) = [~ArH~(sln))'/RT2]1y/(l+ K)' (1) Hence, by fitting the dependence of C,,(T) on temperature, we obtain the van't Hoff enthalpy term, ArHZ(sln).The maximum in C,,(T) occurs at the maximum Tm,vH. However, in the study reported here, the procedures were not straight- forward. In the case of DDP(aq), the concentration of DDP was known when expressed in (mol monomer) dm-3. However, the phenomena described below are properties of DDP vesicles. In practice, the data showed that the states X(aq) and Y(aq) describe a group of DDP monomers which change their form co-operatively. The number of monomers in each group was called the patch number. In other words, we tentatively characterise the groups by patch numbers and refer to each group as a patch. Hence, in fitting the depend- ence of SC, on T to eqn. (l),three parameters are used in the least-squares analysis ; i.e.patch number, Ar H$(sln) and Tm.vH * The equilibrium constant K in eqn. (1) describes the equilibrium between the two states, gel and liquid crystal. The curve formed by the dependence of C, on T has a maximum at Tm,intand the area defined by the bell-shaped 1906 0.02 c I Y-(D go -0.02 (e 1 I I I 20 40 60 80 TJT Fig. 1 Dependence on temperature of the differential heat capacity, JC,,, of DDP(aq) as a function of DDP concentration [DDP] = (a) mol dm- '. (b)2 x (c) 1 x lop3,(d) 5 x and (e)1 x8.4 x plot yields the integrated enthalpy term A, Hgt. Confidence in the analysis of each set of data was gained because in the cases reported below A, HEt and A, Hz are in agreement. Results As a preliminary to the results reported here, differential scans were recorded for solutions prepared using a range of protocols.We observed that scans recorded for solutions pre- pared using the ethanol-injection method' were not repro- ducible. Here we simply report details of the scans for solutions prepared using the protocol reported above. In detail, we examined the traces as a function of DDP(aq) con- centration (Fig. 1). With decrease in concentration, the intensity of the extremum declined. No extremum was observed for solutions containing <1 x (mol monomer) mol-' indicating that the concentration of vesicles was too small to be observed via a change in heat capacity as a func- tion of temperature. The scan pattern for solutions contain- ing 2.0 x mol dm-3 DDP was reproducible over four repeated scans for the same solution.Evidence of a longer- term change was shown by additional extrema after standing Y 0.041 TpC Fig. 2 Dependence on temperature of the differential heat capacity, JC,, of DDP [aq; 8.4 x (monomer mol) dm-3]. (a),(b),(c)and (d) recorded after cooling solutions to 15 "C and scanning to 90 "C. Plot (e) was recorded 10 h after scan (4. J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 at 15°C for 11 h. With increase in [DDP], T, decreases slightly. Nevertheless, there. is only one important extremum in the range 15-90 "C which for a given solution was repro- ducible over a series of repeat scans; Fig. 2. Discussion The striking feature in the DSC scans for the DDP(aq) ves- icles is the extremum near 35 "C which is assigned to a gel-to- liquid-crystal transition.In other words, the n-dodecyl chains within the bilayer 'melt' and gain local freed~m.~ In the present context, use of the term 'melt' is consistent with a description of the process responsible for the extremum as a phase transition. As for other solid +liqiud phase transitions, the melting temperature for surfactants containing the same head group, depends on chain length and chain branching within the amphiphile. In these terms, the breadth of the transition recorded by DSC is attributable to a distribution of vesicle sizes associated with a given preparative meth~d.~ While the above description of the processes responsible for the DSC extrema is attractive, it is difficult to account for the reproducibility of the total scan maximum together with 'band' shape and width.In the same way, it is diflicult to account for the reproducibility of the traces through several heat-cool-heat cycles if the breadth were somehow a for- tuitous consequence of the size and shape distribution amongst vesicles in a given sample. The term melting also seems inappropriate when, according to fluorescence polari- sation studies,' some vesicle transitions occur over a range of 50°C; e.g. vesicles formed in aqueous solution from (C1,H,,O),PO,-Naf. The arguments presented above prompt us to consider other descriptions, particularly one similar to that used in the context of enzyme denaturation. In the latter case, DSC traces often show extrema at a temperature characteristic of the enzyme.13 This temperature indicates a change in the bio- macromolecule from active to inactive forms.Moreover, this change involves a change in organisation throughout large domains within the enzyme. The change in structure can often be de~cribed'~in terms of two equilibrium states leading to an equation of the form shown in eqn. (1). We suggest that this description can be used to account for the DSC scans reported here for DDP vesicles in aqueous solu- tion. Moreover, this approach builds on an intuitively attractive chemical model. In adapting eqn. (1) to the DSC scans we introduced the concept of a patch number. Thus the transition gel -+ liquid crystal does not involve the simultaneous and coupled motion of all DDP monomers in a vesicle, a number much smaller than the aggregation number, e.g.50000 for vesicles formed by dioctadecylammonium chloride (see ref. 8 for details). In other words, the patch number calculated on the basis of eqn. (1)is not the total number of DDP monomers in each vesicle. Rather this number reflects the number of mol- ecules in the DDP bilayers which, acting as a single unit, gain local mobility at T, together with increasing freedom of the phosphate head groups. The derived enthalpy parameters reflect the change in enthalpy for each group of DDP mono- mers. In Fig. 3 we compare the dependence of C,, recorded near 35°C and the best-fit dependence using eqn.(1). In the example shown, agreement between calculated and observed dependences of molar heat capacity on temperature required a patch number of 168. The derived parameters for differen- tial scans produced by solutions of different DDP(aq) concen- tration are recorded in Table 1. The agreement between ArHEt and ArH$ indicates that the analysis is on the right track. With decrease in [DDP], the patch number increases, J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 Table 1 Derived parameters for DDP in aqueous solution as a function of DDP concentration [DDP]/10-3 (monomer mol) dmP3 Tm,intl°C ArHZJkcal (patch mol)-' Tl."H/OC ArH:&cal (patch mo1)- n 8.42 34.8 f0.2 654 3 34.9 f0.2 653 5 168 2.0 35.1 +_ 0.1 642 f21 35.0 f0.1 660f3 250 1 .o 35.3 f0.1 811 f36 35.3 & 0.1 838 f22 500 0.5 35.7 f0.1 734 f39 35.8 f0.1 742 f18 467 patches are loosely coupled. Hence, rapid diffusion of water and other solutes across the bilayer would occur at the joins 600 of the patches.r I Y We thank the University of Leicester for a travel grant to r I 0 400 M.J.B. and SERC for their support under the Molecular -E Recognition Initiative. m 5 CIEA 200 References 1 A. Wagenaar, L. A. M. Rupert, J. B. F. N. Engberts and D. Hoekstra, J. Org. Chem., 1989,54,2638.C 2 J. H. Fendler, Acc. Chem. Res., 1980, 13, 7. 3 T. Kunitake, Angew. Chem., Znt. Ed. Engl., 1992,31, 709. TI"C 4 A. M. Carmona-Ribeiro, Chem. SOC. Rev., 1992,21,209. Fig.3 Dependence of molar heat capacity, C,, ,on temperature for 5 T. A. A. Fonteyn, J. B. F. N. Engberts and D. Hoekstra, Cell and DDP [aq; 8.4 x (monomer mol) dm-3]. Comparison between Model Membrane Interactions, ed. S. Ohki, Plenum Press, New observed dependence (-) and the dependence calculated using York, 1991, p. 215. eqn. (1) (---) together with a patch number of 168. 6 T. A. A. Fonteyn, D. Hoekstra and J. B. F. N. Engberts, J. Am. Chem. SOC., 1990,112,8870. 7 A. M. Carmona-Ribeiro and S. Hix, J. Phys. Chem., 1991, 95, tending towards a maximum near 500. Another pattern 1812. shows that as [DDP] decreases so the molar isobaric heat 8 M. J. Blandamer, B. Briggs, P. M. Cullis, J. A. Green, M. capacity at T, [eqn. (l)] decreases. The dependence of patch Waters, G.Soldi, J. B. F. N. Engberts and D. Hoekstra, J. Chem. SOC.,Faraday Trans., 1992,88,3431.number on monomer concentration indicates that with 9 R. A. Robinson and R. H. Stokes, Electrolyte Solutions, Butter-decrease in concentrations the mean vesicle size decreases. In worths, London, 2nd edn., 1959. the limit the vesicles are sufficiently small that the total 10 L. R. De Young and K. A. Dill, J. Phys. Chem., 1990,94,801. bilayer system melts in a single co-operative transition. 11 L. A. M. Rupert, J. B. F. N. Engberts and D. Hoekstra, Biochem-Expressed in terms of the enthalpy change of each istry, 1988, 27, 8232. monomer unit the changes in enthalpy are quite modest. The 12 M. J. Blandamer, B. Briggs, P. M.Cullis and G. Eaton, J. Chem. Soc., Faraday Trans., 1991,87, 1169. concept of a patch number has wider implications. Many 13 J. M. Sturtevant, Annu. Rev. Phys. Chem., 1987,38,463.models of vesicles describe a uniform bilayer in which the amphiphilic molecules (anions for DDP) are packed with hydrophobic alkyl chains in close contact. In terms of the patch model, the bilayer resembles a quilt in which the Paper 41009245 ;Received 15th February, 1994