Impurities in Commercial Phytantriol Significantly Alter Its Lyotropic

Jun 4, 2008 - Solubilization of Vitamin E into HII LLC Mesophase in the Presence and in the Absence of Vitamin C. Liron Bitan-Cherbakovsky , Idit Yuli...
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6998

Langmuir 2008, 24, 6998-7003

Impurities in Commercial Phytantriol Significantly Alter Its Lyotropic Liquid-Crystalline Phase Behavior Yao-Da Dong,† Aurelia W. Dong,† Ian Larson,† Michael Rappolt,‡ Heinz Amenitsch,‡ Tracey Hanley,§ and Ben J. Boyd*,† Department of Pharmaceutics, Victoria College of Pharmacy, Monash UniVersity, ParkVille, VIC 3052, Australia, Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences, Schmiedlstrasse 6, A8042, Graz, Austria, Bragg Institute, Australian Nuclear Science and Technology Organisation, Menai, NSW 2234, Australia ReceiVed February 20, 2008. ReVised Manuscript ReceiVed April 8, 2008 The lyotropic liquid-crystalline phase behavior of phytantriol is receiving increasing interest in the literature as a result of similarities with glyceryl monooleate, despite its very different molecular structure. Some differences in the phase-transition temperature for the bicontinuous cubic to reverse hexagonal phase have been reported in the literature. In this study, we have investigated the influence that the commercial source and hence the purity has on the lyotropic phase behavior of phytantriol. Suppression of the phase-transition temperatures (by up to 15 °C for the bicontinuous cubic to reverse hexagonal phase transition) is apparent with lower-purity phytantriol. In addition, the composition boundaries were also found to depend significantly on the source and purity of phytantriol, with the bicontinuous cubic phase + excess water boundary occurring at a water content above that reported previously (i.e., >5% higher). Both the temperature and compositional changes in phase boundaries have significant implications on the use of these materials and highlight the impact that subtle levels of impurities can play in the phase behavior of these types of materials.

Phytantriol (3,7,11,15-tetramethylhexadecane-1,2,3-triol), illustrated in Figure 1A, is a lipid that has been used in cosmetics and is available commercially in relatively high purity. Functionally, it improves moisture retention and acts as a penetration promoter,2 leading to recent interest in transdermal drug delivery.3,4 However, the main reason for the recent interest in phytantriol has been the similarity between the lyotropic phase behavior of phytantriol and glyceryl monooleate,5 in particular, the fact that phytantriol also forms a bicontinuous cubic phase (QII) in excess water at ambient temperature and an inverse hexagonal phase (HII) in excess water at higher temperatures.6 In drug delivery applications, the phase structure has a dramatic effect on drug release from the matrix, with the HII phase displaying a slower release than QII7 by virtue of the generally small water channels, and hence there is the potential to change the drug release rate in situ through changes in the phase structure. One feature of the glyceryl monooleate + water phase diagram is that the QII + H2O f HII + H2O transition temperature is relatively high at around 90 °C, whereas that of phytantriol was reported to be approximately 44 °C (ref 6, also illustrated in Figure 7B). This stimulated our interest in phytantriol lyotropic mesophases as a material to “switch” the drug release rate using temperature changes close to physiological temperature (37 °C). Consequently, we further investigated the lyotropic

phase behavior of phytantriol and found a much higher QII + H2O f HII + H2O transition temperature (around 65 °C).8 We also demonstrated the rather dramatic effect that a model lipophilic impurity (vitamin E acetate) has on the transition temperatures in the pseudo-phytantriol + water system, as illustrated in the reproduced partial phytantriol-vitamin E acetate-water phase diagram in Figure 1B; less than 5% vitamin E acetate suppressed the QII + H2O f HII + H2O transition temperature from around 65 °C to below ambient temperature. Polyzos et al. have reported the QII + H2O f HII + H2O transition for phytantriol to occur at 54 °C.9 Hence, we hypothesize that the differences between reported phase behavior in the phytantriol + water system may be due to differing impurity profiles between phytantriol obtained from different commercial suppliers. To test this hypothesis, we further investigated the differences in the lyotropic phase behavior of phytantriol with similar nominal purity from two different commercial sources, BASF and Roche, by differential scanning calorimetry (DSC), crossed polarized light microscopy (CPLM), and small-angle X-ray scattering (SAXS). Differences in chemical composition were investigated by mass spectrometry (MS) and liquid chromatography-mass spectrometry (LC-MS). Finally, we present an alternate phytantriol + water phase diagram for comparison to the previously published diagram prepared with material from a different supplier that may more accurately reflect the lyotropic phase behavior of pure phytantriol.

* Corresponding author. Tel: +61 3 99039112. Fax: +61 3 99039583. E-mail: [email protected]. † Monash University. ‡ Austrian Academy of Sciences. § Australian Nuclear Science and Technology Organisation.

Materials. Phytantriol was purchased from Roche (GrenzachWyhlen, Germany) with a nominal purity of >96.6% (from product specifications by gas chromatography) and BASF (Ludwigshafen,

Introduction

(1) Wagner, E. Parfuem. Kosmet. 1994, 75, 260, 263-4–266-7. (2) Erlemann, G.; Merkle, R. Seifen, Oele, Fette, Wachse 1991, 117, 379–84. (3) Richert, S.; Schrader, A.; Schrader, K. Int. J. Cosmet. Sci. 2003, 25, 5–13. (4) Bender, J.; Ericson, M. B.; Merclin, N.; Iani, V.; Rosen, A.; Engstrom, S.; Moan, J. J. Controlled Release 2005, 106, 350–360. (5) Briggs, J.; Chung, H.; Caffrey, M. J. Phys. II 1996, 6, 723–751. (6) Barauskas, J.; Landh, T. Langmuir 2003, 19, 9562–9565.

Materials and Methods

(7) Boyd, B. J.; Whittaker, D. V.; Khoo, S.-M.; Davey, G. Int. J. Pharm. 2006, 309, 218–226. (8) Dong, Y. D.; Larson, I.; Hanley, T.; Boyd, B. J. Langmuir 2006, 22, 9512– 9518. (9) Polyzos, A.; Alderton, M. R.; Dawson, R. M.; Hartley, P. G. Bioconjugate Chem. 2007, 18, 1442–1449.

10.1021/la8005579 CCC: $40.75  2008 American Chemical Society Published on Web 06/04/2008

Impurities in Commercial Phytantriol

Figure 1. (A) Chemical structure of phytantriol and (B) reproduction of the partial phytantriol-vitamin E acetate-water phase diagram from Dong et al.8

Germany) with a purity of >95%. Milli-Q-grade water (0.05 µS cm-1 at 25 °C) purified through a Millipore system (Sydney, Australia) was used throughout this study. Preparation of Binary Phytantriol/Water Systems (Bulk Phase). Phytantriol from different suppliers was used to prepare the bulk phase in excess water by the method of de Campo et al.10 using repeated cycles of heating, vortex mixing, and centrifugation in HPLC vials for comparison using DSC, CPLM, and SAXS. Samples for SAXS in excess water were then heated and vortex mixed once more to form the low-viscosity L2 phase mixture and injected into glass capillaries (Charles Supper, Natick, MA) and left in a horizontal position for at least 7 days at room temperature to allow for equilibration. Samples for the phase diagram determination were prepared by accurately weighing phytantriol (Roche) and water separately into ampoules that were flame sealed and then treated according to de Campo et al.10 to achieve homogeneous mixing over 21 days. The ampoules were allowed to equilibrate at room temperature for 7 days undisturbed before transfer to bulk phase holders for synchrotron SAXS measurements described below. Small-Angle X-ray Scattering (SAXS) Measurements. For comparison of transition temperatures by SAXS for samples in capillaries, a laboratory-based SAXS system was used (Bruker Nanostar) as described previously.8 The temperature was controlled by use of a Peltier system that was accurate to (0.1 °C. Samples were pre-equilibrated at a particular measurement temperature for 120 min before making a 30 min exposure under vacuum. For the determination of the phytantriol phase diagram, samples were loaded into a custom-built bulk phase holder in which the sample was sandwiched between two thin polymer foil windows in a stainless steel holder and sealed with O-rings. The holder was then inserted into a thermostatted brass block (temperature accurate to (0.1 °C) and placed in the beam path of the SAXS beamline at the Elettra synchrotron (Trieste, Italy) with a radiation wavelength of 1.54 Å.11,12 The holder was allowed to equilibrate at 20 °C for 30 min before acquiring the first scattering pattern and 10 min after temperature equilibration at subsequently increased temperatures. (It should be noted that during the equilibration period the shift in peak position was followed during short (10 s) acquisitions and that shifts in peak position were generally complete within 1 to 2 min after changing the temperature.) Patterns used in phase indexing were acquired for 120 s. The 1D position-sensitive detector for SAXS covered the q range (q ) 4π sin(θ)/λ) of interest from 0.03 to 0.5 Å-1, and the angular calibration was performed with silver behenate (CH3(CH2)20-COOAg) with a d spacing of 58.38 Å.13 Phase identification for all SAXS measurements was made by correlation of the peak positions in the intensity versus q-scattering pattern, with expected peak positions for the diamond (Pn3m) and gyroid (Ia3d) cubic phases and for the HII phase. The lamellar phase was identified by a single sharp peak (the q range of the detector (10) de Campo, L.; Yaghmur, A.; Sagalowicz, L.; Leser, M. E.; Watzke, H.; Glatter, O. Langmuir 2004, 20, 5254–5261. (11) Bernstorff, S.; Amenitsch, H.; Laggner, P. J. Synchrotron Radiat. 1998, 5, 1215–1221. (12) Amenitsch, H.; Rappolt, M.; Kriechbaum, M.; Mio, H.; Laggner, P.; Bernstorff, S. J. Synchrotron Radiat. 1998, 5, 506–508. (13) Huang, T. C.; Toraya, H.; Blanton, T. N.; Wu, Y. J. Appl. Crystallogr. 1993, 26, 180–184.

Langmuir, Vol. 24, No. 13, 2008 6999 was such that only one peak was evident in these studies), whereas the L2 phase was identified by a broad, diffuse scattering maximum. Lattice parameter values were determined by the slopes of the d spacing versus reciprocal Miller index plots for the relevant mesophase as previously described.8 Cross-Polarized Light Microscopy (CPLM). Cross-polarized light microscopy was performed using a Zeiss Axiolab E microscope (Carl Zeiss, Melbourne, Australia) fitted with cross-polarizing filters as described previously.8 A magnification of 150× was used to observe the texture of the sample at the water/lipid interface and was compared to those reported by Rosevear14,15 in order to assign the appropriate mesophases. The samples were heated from room temperature to 80 °C at a rate of 1 °C/min using a Linkam HFS 91 heating stage and a TP-93 temperature programmer (Linkam, Surrey, England). Differential Scanning Calorimetry (DSC). DSC measurements were performed in a similar manner to those reported by Abe and Takahashi.16 The equilibrated phytantriol + water sample (10-20 mg) was accurately weighed into an aluminum pan and hermetically sealed before being placed into a Perkin Elmer DSC7 calorimeter, and thermograms were obtained at a scan rate of 5 °C/min under nitrogen. Electrospray Mass Spectroscopy (ESI-MS). Initial purity investigations were conducted using a Micromass Platform II ESI/ APCI quadrupole mass spectrometer (Manchester, U.K.) in negative ion mode. The phytantriol samples from different suppliers were injected directly as a solution in an acetonitrile (ACN)/water 50:50 v/v mixed solvent system, with the scan range set to mass/charge (m/z) 50-1050. The samples were analyzed using cone voltages of 20, 70, and 100 V. Liquid Chromatography-Mass Spectroscopy (LC-MS). Further purity investigations were conducted by LC-MS on a system comprising a Shimadzu CBM-20A system controller, a SIL-20AC autoinjector, and an LC-20AD pump (Shimadzu Scientific Instruments, Sydney, Australia). A Shimadzu LCMS 2010 single quadrupole mass spectrometer (Shimadzu Scientific Instruments, Sydney, Australia) with an electrospray interface in negative mode was used for selective ion monitoring for m/z ) 375.3 and 391.3 corresponding to the major and minor ions observed in the ESI-MS spectrum for the Roche phytantriol sample, respectively. Phytantriol was dissolved in ACN/water 50:50 at 5 µg/mL, and an injection volume of 20 µL eluted at 0.2 mL/min on a Gemini C18 3 µm, 50 mm × 2 mm column (Phenomenex, Sydney, Australia) using a gradient elution profile (solvent A ) 5% ACN + 95% 0.1% ammonium formate, solvent B ) 95% ACN + 5% 0.1% ammonium formate; gradient program: t ) 0 min, 10% solvent B; t ) 0.5-5 min ramp for 10-100% solvent B; t ) 5-5.5 min ramp for 100% solvent B; t ) 5.5-6 min ramp for 100-10% solvent B; t ) 6 to 10 min, solvent B ) 10%).

Results Comparison of the Lyotropic Phase Behavior between Different Sources of Phytantriol. Both phytantriol samples in contact with excess water displayed similar temperatures for the conversion from the nonbirefringent, viscous cubic phase (QII) to the birefringent reverse hexagonal (HII) phase by CPLM, with the BASF sample having a slightly narrower range of coexistence of the two phases (59-60 °C) than the Roche sample (59-61 °C). For the BASF-source phytantriol in excess water, the conversion from the birefringent, viscous HII phase to the nonbirefringent, low-viscosity L2 phase occurred between 68 and 69 °C. In comparison, the Roche-source phytantriol in excess water converted from HII to L2 at 65-66 °C. When the phase transitions were investigated using DSC, the results showed similar behavior to that observed by CPLM (Figure 2). The onset of the first endotherm representing the QII to HII (14) Rosevear, F. B. J. Soc. Cosmet. Chem. 1968, 19, 581–594. (15) Rosevear, F. B. J. Am. Oil Chem. Soc. 1954, 31, 628–639. (16) Abe, S.; Takahashi, H. J. Appl. Crystallogr. 2003, 36, 515–519.

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Figure 2. DSC thermogram of phytantriol obtained from Roche and BASF, both in excess water. The DSC scan rate was 5 °C/min.

Figure 4. Comparison of SAXS patterns with increasing temperature at 65, 70, and 75 °C, covering the region of HII phase formation, for (A) Roche phytantriol and (B) BASF phytantriol in excess water. Bragg peaks are indexed as hkl (e.g., 111) with D denoting reflections for the Pn3m (diamond) space group of the QII phase and H denoting reflections for the HII phase.

Figure 3. Temperature dependence of the lattice parameter (a) for the phytantriol-water system. Closed symbols indicate data obtained using BASF phytantriol, and open symbols indicate Roche as the source of the phytantriol. Circles indicate QPn3m + excess water; squares indicate HII* + excess water; and triangles indicate L2 + excess water. *For the Roche sample, a pure HII + water region was not observed, and the white square represents a region of coexistence of HII + excess water with the QII and L2 phases.

conversion occurred at 56.9 ( 0.1 °C for the Roche-source phytantriol and at 56.6 ( 0.2 °C for the BASF sample. The first endotherm peak for the BASF phytantriol was also sharper than that of the Roche phytantriol, indicating a more narrow temperature range for the QII to HII coexistence, consistent with the CPLM results. The phytantriol from BASF showed a significantly higher onset for the second endotherm of 65.5 ( 0.2 °C, which likely represents the HII to L2 phase conversion, compared to that of the Roche sample that was at 61.4 ( 0.3 °C. Small angle X-ray scattering of the samples conducted using temperature control and an increasing temperature profile also supported the findings from CPLM and DSC. The scattering profiles for both phytantriol samples in excess water showed the existence of the Pn3m diamond cubic phase in the temperature range of 25-65 °C and the L2 phase at temperatures g75 °C (Figure 4). The strong reflection in the D211 plane in Figure 4B is likely due to the alignment of a large crystallite in the sample in the incoming beam. At 70 °C, the scattering pattern from the Roche phytantriol indicated the coexistence of the QII and HII phases, and the broadening of the peaks also indicated the likely commencement of the transition to the L2 phase. In contrast, the patterns for the BASF phytantriol clearly showed that only the HII phase was present at 70 °C. The overlaying lattice parameter data extracted from the scattering obtained from SAXS patterns with increasing temperature (Figure 3) indicated that the QII and HII phase structures formed by both Roche and BASF phytantriol samples had similar water contents, albeit with the transitions between them occurring at different temperatures. The decrease in lattice parameter with increasing temperature is typical of these systems and is mainly due to increasing trans-gauche

isomerization of the hydrocarbon chains, which leads to an overall shortening of the chains.8 Phytantriol Purity. ESI-MS electropherograms in negative ion mode indicated numerous m/z peaks for the Roche phytantriol that were absent in the BASF phytantriol, indicating the higher purity of the BASF phytantriol sample. In particular, the peak at m/z ) 391 was prominent in the Roche sample but absent in the BASF sample. The peak at m/z ) 375 was believed to be due to phytantriol; it has been reported previously that lipids can form the formate adduct ([M + COOH]-) in negative ion mode mass spectrometry,17 and considering that the molecular weight of phytantriol is 330 g/mol, the formate adduct would be expected to have an m/z of 375. Hence, although there is no direct proof, there is strong evidence that the m/z ) 375 ion is directly attributable to phytantriol. Furthermore, the BASF product specification data sheet for phytantriol states that the tetrahydroxy derivative of tetramethyl-hexadecane is a potential impurity in phytantriol; the formate adduct of this compound would be expected to show an m/z of 391, providing strong evidence that phytantriol produces m/z ) 375, whereas the tetraol derivative produces the species at m/z ) 391. Further supporting evidence is the fact that the m/z ) 391 species eluted earlier (3 min) from the reverse-phase HPLC column (B) than did the m/z ) 375 species (A) (4.15 min), a result that is expected for a more hydrophilic compound. It was also noted that increasing phytantriol concentration led to greater integrated area for the peaks in LC-MS (not shown), indicating that both ions arose directly from the Roche sample. We cannot predict the relative proportion of the two components in the mixture because of the potential for differences in ionization. However, considering their very similar structures, the ionization is likely to be similar, in which case the m/z ) 391 species would be only a minor contributor to the overall composition of the Roche sample. Alternate Phytantriol-Water Phase Diagram. In light of the effect that relatively small quantities of impurities can have on the lyotropic phase behavior of phytantriol and the fact that our previous data in excess water8 and additional findings here (17) Sommer, U.; Herscovitz, H.; Welty, F. K.; Costello, C. E. J. Lipid Res. 2006, 47, 804–814.

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Figure 5. ESI-MS spectra for (A-C) Roche and (D-F) BASF phytantriol at varying cone voltages (A , D ) 100 V; B, E ) 70 V; C, F ) 20 V). The ion detected at m/z ) 391 in Roche phytantriol is not present in BASF phytantriol.

Figure 6. LC-MS investigation of phytantriol composition. The Roche sample provided peaks corresponding to the two ions found in the ESI spectrum in Figure 5, with ions detected at both m/z ) 375 and 391 (A and B, respectively). Note the earlier retention time for the m/z ) 391 peak, indicating its greater hydrophilicity. For the BASF sample, only one peak was found at m/z ) 375, and no peaks were found when selecting for m/z ) 391 or in the total ion count (TIC) chromatogram (C-E).

have indicated a substantially higher QII to HII transition temperature, we felt it useful to revisit the phytantriol-water

equilibrium phase diagram to determine whether any other parameters may also be susceptible to the source and purity of phytantriol. The phase diagram produced using synchrotron SAXS, illustrated in Figure 7A, was in broad agreement with that reported previously by Barauskas and Landh;6 however, there were significant differences in a number of features. Whereas the QII Pn3m to HII to L2 transitions with increasing temperature at high water content were still evident, the QII Pn3m to HII transition temperature (marked 1 in Figure 7B) was found to be significantly higher than that previously reported. The QII Pn3m persisted up to 58 °C, and HII existed only from 60-62 °C and transformed completely to the L2 phase at 64 °C. Thus, the range of existence of the HII phase was very narrow in comparison to that in Figure 7B. The phase progression at low temperature (20 °C) was also the same as previously reported. The progression from L2 to LR to QII Ia3d to QII Pn3m with increasing water content was observed. However, in the previously reported phase diagram, the boundary between the QII Ia3d + QII Pn3m transition region with QII Pn3m (marked 2 in Figure 7B) was at 25% water, and the QII Pn3m to QII Pn3m + excess water boundary was at approximately 29% water. In this study, it was found that these transitions occur at a higher water content, with the former transition occurring at approximately 33% water. In fact, the difference in composition at the excess water boundary between the two studies led to a design for this study with samples containing up to 34% water in the belief that the excess water boundary would be encountered at a lower composition. In fact, at 20 °C it was only just possible to delineate the pure QII Pn3m phase from the QII Ia3d + QII Pn3m transition region using this sample regime. Therefore, it is likely that a higher water content would be required to access the QII Pn3m + excess water boundary. Swelling studies conducted

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recently by Rizwan et al.18 in which water uptake into phytantriol was determined gravimetrically have indicated that the QII Pn3m f QII Pn3m + excess water boundary occurs at approximately 36% w/w water. Thus, it appears that the important phase boundary with excess water (from an application and cubosome preparation perspective) using this source of phytantriol may actually be off scale on the previously reported phase diagram in Figure 7B, which reported compositions only up to 35% water. In addition, the QII Ia3d + QII Pn3m transition region (indicated by 3 in Figure 7B) appeared to be a nominal transition region in the previously published phase diagram but was found in this study to occupy a more substantial proportion of the phase diagram. This phase transition was found to span a region of 15% w/w water by composition and a range of 40 °C in temperature and hence is a more dominant feature in this alternative phase diagram. Lastly, it was also noted that the melting temperature of the lamellar to L2 phase at approximately 40 °C is also higher than that reported previously at 35 °C.

Discussion The issue of surfactant purity impacting the phase behavior is important from the perspective of not only gaining an understanding of the true phase behavior at the molecular purity level but also (i) gaining insight into and (ii) optimizing the behavior of commercially manufactured complex mixtures. Surfactant purity can strongly impact the lyotropic liquidcrystalline behavior,19,20 and phytantriol appears to be one lipid that is particularly sensitive to low levels of impurities altering its phase behavior in water.8 The nature of the impact of impurities on the bicontinuous cubic phase to hexagonal phase transition depends on the influence of the impurity on the spontaneous curvature at the lipid-water interface and is generally understood using the packing parameter concept.21 Interfacially active impurities that promote bilayer curvature are likely to stabilize the bicontinuous cubic phase structure against a transition to the reverse hexagonal phase, leading to higher temperatures for the QII to HII transition. Cholesterol has been shown to have this effect on the reverse hexagonal phase formed by 1-palmitoyl-2-oleoyl-phosphatidylethanolamine.22 Conversely, interfacially active compounds with increased hydrocarbon chain volume (in double-chained nonionic amphiphiles such as dioleoyl-phosphatidylcholine) will promote the formation of the reverse hexagonal phase at lower temperature. In addition, noninterfacially active hydrophobic compounds (such as oils23 and vitamin E acetate mentioned earlier8) may also promote the formation of the reverse hexagonal phase at lower temperatures by occupying an energetically unfavorable “void” in the space between hexagonally packed cylinders, whose formation is thought to increase the temperature of the QII to HII transition.24,25 (18) Rizwan, S. B.; Boyd, B. J.; Rades, T.; Hook, S. Characterisation, Swelling and Release from Liquid Crystalline Systems of Phytantriol and Glyceryl Monooleate as Platforms for Controlled Release of Bioactives. Proceedings of the Controlled Release Society; New York, 2008. (19) Laughlin, R. G. The Aqueous Phase BehaViour of Surfactants. Academic Press: London, 1994; p 558. (20) Boyd, B. J.; Drummond, C. J.; Krodkiewska, I.; Weerawardena, A.; Furlong, D. N.; Grieser, F. Langmuir 2001, 17, 6100–6107. (21) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525–1568. (22) Wang, X. Y.; Quinn, P. J. Biochim. Biophys. Acta 2002, 1564, 66–72. (23) Yaghmur, A.; de Campo, L.; Sagalowicz, L.; Leser, M. E.; Glatter, O. Langmuir 2005, 21, 569–577. (24) Gruner, S. M.; Tate, M. W.; Kirk, G. L.; So, P. T. C.; Turner, D. C.; Keane, D. T.; Tilcock, C. P. S.; Cullis, P. R. Biochemistry 1988, 27, 2853–2866. (25) Templer, R. H.; Seddon, J. M.; Duesing, P. M.; Winter, R.; Erbes, J. J. Phys. Chem. B 1998, 102, 7262–7271.

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The differences in behavior between the two commercially obtained phytantriol samples were relatively subtle but illustrate how small compositional differences can influence lyotropic behavior. The lower-purity Roche sample had lower transition temperatures, which is a result anticipated from our previous work with phytantriol. Importantly, the magnitude of the QII Pn3m to HII transition temperatures (approximately 65 °C by SAXS but slightly lower by CPLM and DSC) was consistently higher than previously reported (44 °C6). This finding supports our earlier studies on the phytantriol system reproduced in Figure 1B in which the transition temperature in the absence of vitamin E acetate was also found to occur at approximately 65 °C. A higher QII Pn3m to HII transition temperature for the phytantriol + excess water system was also reported by Polyzos et al. at 54 °C, who used phytantriol obtained from Aldrich.9 The higher transition temperature means that the lyotropic phase behavior is more similar to that of glyceryl monooleate than previously believed, for which the same transition occurs at 90 °C. The previously reported transition for phytantriol at 44 °C was reasonably close to physiological temperatures, suggesting the possibility of changing phase behavior in vivo to control drug release rates. Although this may still be achievable, it would require greater-than-anticipated adjustments to the phase behavior using additives. Unfortunately, we were unable to obtain a sample of phytantriol from the same source as that used by Barauskas and Landh6 (from Kuraray Co. Ltd., Japan) for phase confirmation and further compositional analysis. However, on the basis of the magnitude of differences between the BASF/Roche samples used in the studies presented here and our previous experience with vitamin E acetate doped into phytantriol (Figure 1B), we consider it highly likely that a difference in the impurity profile between the commercial samples is responsible for the variation in the observed results. On the basis of the findings reported here, it would appear that the tetraol impurity may be acting more like an HII stabilizing amphiphile (i.e., which has a similar effect on phase behavior as vitamin E acetate in lowering the QII to HII transition). It could be anticipated from the tetraol structure (slightly more hydrophilic than phytantriol) that the opposite effect should occur, although it can often be difficult to predict the impact of a new amphiphile on the phase structure exactly. It also cannot be excluded that an as yet unidentified second impurity has a strong impact on the structure despite its presence in very low quantities. Future studies on the synthesis of the pure tetraol and an evaluation of its impact at increasing concentration should shed more light on its relative impact on phytantriol behavior. However, if we accept that vitamin E acetate induces a similar effect in suppressing the QII Pn3m to HII transition as the tetraol impurity in commercial phytantriol samples, the data reproduced in Figure 1B suggest that for a reduction of the transition temperature to 44 °C approximately 3% of the impurity is likely to be present. Although this is a relatively small quantity for a commercially prepared product, it highlights the need for an awareness of purity levels in materials in such studies of lyotropic phase behavior. Having commented on the issue of purity and in light of the apparently greater purity of the BASF material compared to the Roche material, it is unfortunate that BASF has apparently discontinued the manufacture of phytantriol as a commercial product. This is the primary reason for our determination of the alternative phytantriol + water phase diagram in Figure 7A using the Roche phytantriol (now manufactured by DSM Nutritional Products). It should also be noted that further purification of the phytantriol used in this study may have yielded an even higher

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Figure 7. (A) Phase diagram for the phytantriol-water system determined by synchrotron SAXS. The Roche phytantriol was utilized in the preparation of this phase diagram. Only data points that define phase boundaries are included. Circles define samples exhibiting the reverse micellar phase (L2), squares represent the lamellar phase (LR), downward facing triangles represent the gyroid bicontinuous cubic phase (QII Ia3d), upward facing triangles represent the diamond bicontinuous cubic phase (QII Pn3m), hexagons represent the reverse hexagonal phase (HII), and open diamonds denote multiphase transition regions. (B) Reproduction of the phytantriol-water phase diagram from Barauskas et al.6 The annotations on B marked 1, 2, and 3 represent particular features of the phase diagram discussed in the text.

temperature for the QII Pn3m to HII transition and is an aim of future studies in our laboratory. Perhaps of greater importance in the application of these materials was the finding that the excess water boundary occurs at significantly greater water content (>35% at 25 °C in Figure 7A compared to