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Temperature Scanning Ultrasonic Velocity Study of Complex Thermal Transformations in Solid Lipid Nanoparticles Tarek Samir Awad,*,† Thrandur Helgason,†,‡ Kristberg Kristbergsson,‡ Jochen Weiss,† Eric Andrew Decker,† and David Julian McClements† Department of Food Science, UniVersity of Massachusetts, 100 Holdsworth Way, Amherst, Massachusetts 01003, and Department of Food Science and Nutrition, UniVersity of Iceland, Hjardarhagi 2-6, ReykjaVik 107, Iceland ReceiVed July 10, 2008. ReVised Manuscript ReceiVed September 8, 2008 The purpose of this study was to determine whether temperature scanning ultrasonic velocity measurements could be used to monitor the complex thermal transitions that occur during the crystallization and melting of triglyceride solid lipid nanoparticles (SLNs). Ultrasonic velocity (u) measurements were compared with differential scanning calorimetry (DSC) measurements on tripalmitin emulsions that were cooled (from 75 to 5 °C) and then heated (from 5 to 75 °C) at 0.3 °C min-1. There was an excellent correspondence between the thermal transitions observed in δ∆u/δT versus temperature curves determined by ultrasound and heat flow versus temperature curves determined by DSC. In particular, both techniques were sensitive to the complex melting behavior of the solidified tripalmitin, which was attributed to the dependence of the melting point of the SLNs on particle size. These studies suggest that temperature scanning ultrasonic velocity measurements may prove to be a useful alternative to conventional DSC techniques for monitoring phase transitions in colloidal systems.
Introduction Solid lipid nanoparticle (SLN) suspensions consist of fully or partially crystalline lipid nanoparticles suspended in an aqueous continuous phase.1-4 SLN suspensions are typically formed by hot-homogenization of a lipid and an aqueous phase together in the presence of a suitable emulsifier.2 Homogenization is carried out at a temperature above the melting point of the lipid phase, and then the resulting nanoemulsion is cooled to a temperature below the crystallization point of the emulsified lipid. SLNs have proven to be a powerful means of delivering poorly watersoluble drugs in the pharmaceutical industry5-7 and can also be used to protect and deliver lipophilic functional agents in the food industry.8-10 SLN suspensions have a number of beneficial attributes as delivery systems: high encapsulation capacity; good chemical and physical stability; good oral bioavailability; and potential for large scale production.2,3,5,7,11 The stability of SLN suspensions to aggregation, as well as their ability to retain and protect encapsulated components during storage, is strongly * To whom correspondence should be addressed. E-mail: tawadjp@ gmail.com. Telephone: 413-577-3344. Fax: 413-545-1262. † University of Massachusetts. ‡ University of Iceland. (1) Muller, R. H.; Mader, K.; Gohla, S. Eur. J. Pharm. Biopharm. 2000, 50, 161–177. (2) Mehnert, W.; Mader, K. AdV. Drug DeliVery ReV. 2001, 47, 165–196. (3) Bummer, P. M. Crit. ReV. Ther. Drug Carrier Syst. 2004, 21, 1–20. (4) Saupe, A.; Wissing, S. A.; Lenk, A.; Schmidt, C.; Muller, R. H. Bio-Med. Mater. Eng. 2005, 15, 393–402. (5) Mu¨ller, R. H.; Mehnert, W.; Lucks, J. S.; Schwarz, C.; zur Mu¨hlen, A.; Weyhers, H.; Freitas, C.; Ru¨hl, D. Eur. J. Pharm. Biopharm. 1995, 41, 62–69. (6) Wissing, S. A.; Kayser, O.; Mu¨ller, R. H. AdV. Drug DeliVery ReV. 2004, 56, 1257–72. (7) Almeida, A. J.; Souto, E. AdV. Drug DeliVery ReV. 2007, 59, 478–490. (8) Awad, T. S.; Helgason, T.; Kristbergsson, K.; Decker, E. A.; Weiss, J.; McClements, D. J. Food Biophys. 2008, 3, 155–162. (9) Helgason, T.; Awad, T. S.; Kristbergsson, K.; McClements, D. J.; Weiss, J. J. Am. Oil Chem. Soc. 2008, 85, 501–511. (10) Weiss, J.; Decker, E. A.; McClements, D. J.; Kristbergsson, K.; Helgason, T.; Awad, T. S. Food Biophys. 2008, 3, 146–154. (11) Mu¨ller, R. H.; Radtke, M.; Wissing, S. A. AdV. Drug DeliVery ReV. 2002, 54(Suppl 1), S131–55.
dependent on the physical state of the lipids.8,9,12-15 It is therefore important to have analytical methods that can monitor melting, crystallization, and polymorphic transitions in SLN suspensions. A variety of analytical methods have previously been employed to follow thermal transitions of emulsified lipids, with the most widely used being differential scanning calorimetry (DSC), X-ray diffraction, electron microscopy, and neutron scattering.16-18 In this study, we examine the possibility of using temperature scanning ultrasonic velocity measurements to monitor thermal transitions in SLN suspensions. Ultrasonic velocity measurements have been used to monitor the crystallization and melting of emulsified lipids for many years.19-25 However, their potential for following the complex thermal transitions observed within solid lipid nanoparticles has not previously been established. The ultrasonic velocity (u) of a material depends on its density (F) and adiabatic compressibility (κ): u ) (Fκ)-1/2.26 The densities and mechanical properties of bulk materials depend on their physical state, and hence, one would expect that the ultrasonic properties of emulsified lipids would depend on the physical state of the lipid particles. In this study, the ultrasonic technique (12) Bunjes, H.; Steiniger, F.; Richter, W. Langmuir 2007, 23, 4005–4011. (13) Bunjes, H.; Westesen, K.; Koch, M. H. J. Int. J. Pharm. 1996, 129, 159– 173. (14) Westesen, K.; Siekmann, B. Int. J. Pharm. 1997, 151, 35–45. (15) Freitas, C.; Mu¨ller, R. H. Eur. J. Pharm. Biopharm. 1999, 47, 125–32. (16) Relkin, P.; Yung, J. M.; Kalnin, D.; Ollivon, M. Food Biophys. 2008, 3, 163–168. (17) Bunjes, H.; Unruh, T. AdV. Drug DeliVery ReV. 2007, 59, 379–402. (18) Kalnin, D.; Quennesson, P.; Artzner, F.; Schafer, O.; Narayanan, T.; Ollivon, M. Trends Colloid Interface Sci. XVII 2004, 126, 139–145. (19) Awad, T. S. Food Res. Int. 2004, 37, 579–586. (20) Dickinson, E.; Goller, M. I.; McClements, D. J.; Peasgood, S.; Povey, M. J. W. J. Chem. Soc., Faraday Trans. 1990, 86, 1147–1148. (21) Hodate, Y.; Ueno, S.; Yano, J.; Katsuragi, T.; Tezuka, Y.; Tagawa, T.; Yoshimoto, N.; Sato, K. Colloids Surf., A 1997, 128, 217–224. (22) McClements, D. J.; Povey, M. J. W.; Dickinson, E. Ultrasonics 1993, 31, 433–437. (23) Martini, S.; Bertoli, C.; Herrera, M. L.; Neeson, I.; Marangoni, A. J. Am. Oil Chem. Soc. 2005, 82, 305–312. (24) Awad, T. S.; Sato, K. Colloids Surf., B 2002, 25, 45–53. (25) Awad, T. S.; Sato, K. J. Am. Oil Chem. Soc. 2001, 78, 837–842. (26) McClements, D. J.; Povey, M. J. W. Ultrasonics 1992, 30, 383–388.
10.1021/la802199p CCC: $40.75 2008 American Chemical Society Published on Web 10/17/2008
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is compared with DSC for studying the melting and crystallization of tripalmitin nanoparticles.
Experimental Details Materials. Tripalmitin was purchased from Fluka (Buchs, Switzerland). Sodium phosphate monobasic and sodium phosphate dibasic were purchased from Fisher Scientific (St. Clair Shores, MI). Sodium azide, sodium dodecyl sulfate (SDS), and corn oil were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO). Omega-3 (ω-3) rich fish oil from Menhaden was obtained from Omega Protein Corp. (Reedville, VA; eicosapentaenoic acid, 10-17%; docosahexenoic acid, 7-12%) and stored at -80 °C before use. All chemicals were used as received. Methods. Nanoparticle Preparation. Tripalmitin nanoparticles were prepared by high pressure melt homogenization. Tripalmitin was melted and held at 75-80 °C for more than 30 min to avoid crystallization during emulsification. Crystalline fish oil was thawed in tap water for 15 min and then mixed with hot tripalmitin (75-80 °C). Tripalmitin (10% (w/w)) or its mixtures with ω-3 were then mixed with a hot buffered surfactant solution (1.5% (w/w) SDS, 4 mM sodium phosphate monobasic, 6 mM sodium phosphate dibasic, 0.02% sodium azide, pH 7.0, 75 °C). The hot mixture was blended for 1 min using a hand-held high speed blender (model SDT-1810, EN Shaft, Tekmar Co., Cincinnati, OH). The resulting coarse premix was then finely dispersed under pressure (9K bar and 10 times) using a microfluidizer (Microfluidics, Newton MA). This produced an emulsion with a monomodal size distribution and a mean particle diameter (Z-average) of 145 nm. The emulsion was then stored in a plastic container at 37 °C in a temperature-controlled room until use. No change in mean particle diameter was observed after 2 month of storage, and the lipid phase did not crystallize, indicating that this emulsion contained liquid droplets that were stable to aggregation. SLN suspensions were prepared by cooling the 10 wt % oil-in-water (O/W) emulsions from 70 to 5 °C at 0.3 °C min-1. Particle Size Determination. Particle size measurement was performed by photon correlation spectroscopy (PCS) using a Malvern Zetasizer NanoZS instrument (Malvern Instruments, Malvern, U.K.). The samples were diluted 100× in buffer solution at 37 °C. PCS gives the mean diameter of the particle population and the polydispersity index (PI) ranging from 0 (monodisperse) to 0.50 (very broad distribution). To monitor the emulsion stability, the particle size distribution was measured at 37 °C for the emulsion soon after preparation and during a period of 2 months. Typical particle size distributions for emulsions containing pure tripalmitin and 75% tripalmitin/25% fish oil are shown in Figure 1. Differential Scanning Calorimetry. A differential scanning calorimeter (Q1000, TA Instruments, New Castle, DE) was used to study thermal transitions. An aliquot of sample (8-10 mg) was placed in a hermetic aluminum pan and sealed, and an empty pan was used as a reference. All the DSC pans were equilibrated at 37 °C prior to adding the sample to avoid lipid crystallization. The pans were loaded into the DSC instrument at 37 °C, and the temperature was then increased to 75 °C. The samples were then cooled from 75 to 5 °C at 0.3 °C min-1 to promote tripalmitin crystallization, and then heated from 5 to 75 °C at 0.3 °C min-1 to promote tripalmitin melting. Samples of bulk tripalmitin with ω-3 were prepared by melting the appropriate weights and mixing them thoroughly with a vortex for a few seconds. A preheated glass pipet was then used to load the samples into the DSC pans. Ultrasonic Measurements. An ultrasonic resonator (Resonic Instruments GmbH, Ditzingen, Germany) was used to measure the ultrasonic velocity of the tripalmitin O/W nanoemulsions at temperatures ranging from 5 to 80 °C. This instrument has a fixed cell path length of 7 mm and a fixed cell volume of 0.2 mL. Samples were degassed and filled into the resonator cells at 37 °C, and the temperature was then increased to 75 °C. Next, the samples were cooled from 75 to 5 °C at 0.3 °C min-1 to promote tripalmitin crystallization and then heated from 5 to 75 °C at 0.3 °C min-1 to promote tripalmitin melting. The ultrasonic data were recorded as
Figure 1. Particle size distributions of 100% tripalmitin (PPP) and 75%/ 25% tripalmitin/fish oil (PPP:Fish Oil) in water emulsions after 0 (a) and 2 (b) months.
the temperature-dependence of the absolute measured ultrasonic velocity (u) or as the difference in the ultrasonic velocity (∆u) between an emulsion and a buffer solution. In addition, we also calculated the temperature differential of the ultrasonic velocity (δ∆u/δT) by averaging a number of points over a temperature range of about 1 °C. Experiments were performed two or three times using freshly prepared samples.
Results and Discussion DSC Monitoring of Thermal Transitions in Bulk Lipids. Initially, we characterized the thermal transitions of bulk tripalmitin and tripalmitin/fish oil mixtures when they were cooled and heated using differential scanning calorimetry (DSC). These experiments were carried out to so that we could compare the thermal behavior of bulk and emulsified tripalmitin under the same conditions. The ultrasonic velocity technique could not be used to study the behavior of the bulk fats due to the large changes in sample density that occur during crystallization, which would have damaged the ultrasonic resonator cell. The thermal behavior of 100% tripalmitin sample was characterized when it was cooled (from 80 to 5 °C) and then heated (from 5 to 80 °C) at 0.3 °C min-1. During the cooling step, a single exothermic peak was observed around 41.3 °C (Figure 2a), which can be attributed to crystallization of the tripalmitin into the metastable R-polymorphic form.27,28 Upon heating, a single endothermic peak was observed at 62.7 °C (Figure 2b), which can be assigned to melting of the stable (27) Kellens, M.; Meeussen, W.; Reynaers, H. Chem. Phys. Lipids 1990, 55, 163–178. (28) Kellens, M.; Meeussen, W.; Riekel, C.; Reynaers, H. Chem. Phys. Lipids 1990, 52, 79–98.
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Figure 2. DSC thermograms of 100% tripalmitin (bottom) and 75%/ 25% tripalmitin/fish oil mixture (top) during cooling (a) and subsequent heating (b) at a constant rate of 0.3 °C min-1.
Figure 3. DSC thermograms of emulsified tripalmitin (bottom) and emulsified 75%/25% tripalmitin/fish oil mixture (top) during cooling (a) and subsequent heating (b) at a constant rate of 0.3 °C min-1.
β-polymorphic form.27,28 The conversion of the R-to-β-polymorphic forms was not observed because of the relatively slow scan rate used.29 A similar result has also been observed with bulk tristearin where a solid-solid transition from the R- to the β-form occurred at slow heating rates.30 The thermal behavior of the tripalmitin/fish oil sample was also characterized when it was cooled (from 80 to 5 °C) and then heated (from 5 to 80 °C) at 0.3 °C min-1. During the cooling step, a broad exothermic peak was observed around 40.4 °C (Figure 2a), which can be attributed to crystallization of the tripalmitin into the metastable R-form.27,28 This peak was broader than that observed for pure tripalmitin, and it occurred at a slightly lower temperature, which can be attributed to the ability of the fish oil to act as a solvent for the tripalmitin.31 One may also notice the appearance of a small shoulder peak at about 38.8 °C, which may be attributed to an R-to-β solid state polymorphic transition, which occurred after the bulk crystallization. Upon heating, a single endothermic peak was observed at 62.0 °C (Figure 2b), which again can be attributed to melting of the β-form. DSC Monitoring of Thermal Transitions in Emulsified Lipids. The thermal behaviors of emulsified tripalmitin and tripalmitin/fish oil mixtures were then measured using DSC to
determine whether they exhibited similar behavior as the bulk oils. The thermal transitions of pure tripalmitin emulsions were determined when they were cooled (from 75 to 5 °C) and then heated (from 5 to 75 °C) at 0.3 °C min-1 (Figure 3). During the cooling step, a small exothermic peak was observed around 31.9 °C and a much larger one at 24.5 °C (Figure 3a). The small peak may be due to a fraction of free tripalmitin or relatively large tripalmitin droplets that crystallized at a higher temperature than the smaller droplets due to the presence of impurities that promoted heterogeneous nucleation.19,32-36 Alternatively, this peak may have been due to crystallization of the surfactant tails at the oil-water interface because previous works have shown that surfactants influence the crystallization and melting behavior of emulsified lipids and possibly alter the crystal structure (polymorphism) of the lipid phase.24,25,34 The large peak at 24.5 °C can be attributed to crystallization of the tripalmitin droplets into the metastable R-form.27,28 The temperature where crystallization of the emulsified tripalmitin was first observed was considerably lower (about 20 °C) than that of the bulk tripalmitin due to supercooling effects, that is, the low probability of finding a catalytic impurity within an individual emulsion droplet.31,37
(29) Aronhime, J.; Sarig, S.; Garti, N. J. Am. Oil Chem. Soc. 1988, 65, 1144– 1150. (30) Oh, J. H.; McCurdy, A. R.; Clark, S.; Swanson, B. G. J. Food Sci. 2002, 67, 2911–2917. (31) Walstra, P. Physical Chemistry of Foods; Marcel Decker: New York, 2003.
(32) McClements, D. J.; Dungan, S. R.; German, J. B.; Simoneau, C.; Kinsella, J. E. J. Food Sci. 1993, 58, 1148. (33) Kaneko, N.; Horie, T.; Ueno, S.; Yano, J.; Katsuragi, T.; Sato, K. J. Cryst. Growth 1999, 197, 263–270. (34) Awad, T. S.; Hamada, Y.; Sato, K. Eur. J. Lipid Sci. Technol. 2001, 103, 735–741. (35) Awad, T. S.; Sato, K. In Physical properties of lipids; Marangoni, A. G., Narine, S., Eds.; Marcel Dekker: New York, 2002; pp 37-62. (36) Gulseren, I.; Coupland, J. N. J. Am. Oil Chem. Soc. 2008, 85, 413–419.
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Upon heating, a complex series of endothermic peaks was observed in the thermogram (Figure 3b), which were quite different from the single endothermic peak observed in the bulk tripalmitin (Figure 2b). Previous studies of the melting behavior of crystalline triglyceride nanoparticles have also reported that a complex series of endothermic peaks occurred upon heating.38 This phenomenon was attributed to the fact that the melting point of the nanoparticles increased with increasing particle size and there was a range of different particle sizes present within the emulsion studied. The same authors showed that this thermal behavior could not be attributed to polymorphic transitions, since the initial nanoparticles were already in the β-polymorphic form. The emulsions used in our study contained a range of different particle sizes (Figure 1), which would account for the fact that melting was observed over a range of temperatures (Figure 3b). An alternative explanation for the complex melting behavior of the tripalmitin nanoparticles is that surface lipids melt at a lower temperature than core lipids.39 The thermal behavior of the 75% tripalmitin/25% fish oil emulsion was also characterized by DSC when it was cooled (from 75 to 5 °C) and then heated (from 5 to 75 °C) at 0.3 °C min-1 (Figure 3). During the cooling step, a single exothermic peak was observed around 21.3 °C (Figure 3a), which can be attributed to crystallization of the tripalmitin into the metastable R-form. The crystallization temperature was slightly below that of the pure tripalmitin, which can be attributed to melting point depression caused by the presence of the liquid fish oil. Upon heating, the thermal behavior of the emulsified tripalmitin was appreciably different in the 100% PPP and 75% PPP/25% fish oil systems (Figure 3b). In addition, there were appreciable differences between the thermal behavior of the bulk (Figure 2b) and emulsified (Figure 3b) tripalmitin/fish oil systems. During heating, two broad endothermic peaks were observed at 38-42 and 50-60 °C in the emulsified tripalmitin/fish oil system (Figure 3b), whereas only a single narrow peak was observed at 57-63 °C in the bulk tripalmitin/fish oil system (Figure 2b). This difference may again be attributed to the fact that the crystallization temperature of a solid lipid particle increases with increasing particle size, so that there will be a range of melting temperatures in a polydisperse system.38 The differences in the melting behavior of the emulsified tripalmitin in the 100% tripalmitin and 75% tripalmitin/25% fish oil systems (Figure 3b) suggest that the impact of the fish oil on the melting behavior of tripalmitin may depend on particle size. Ultrasonic Monitoring of Thermal Transitions in Emulsified Lipids. The purpose of these experiments was to ascertain whether temperature scanning ultrasonic velocity measurements were sensitive to the various thermal transitions observed in emulsified tripalmitin using DSC. Initially, the temperaturedependence of the ultrasonic velocity of pure corn oil and pure buffer solution was measured (Figure 4) so as to aid in the interpretation of the subsequent ultrasonic velocity measurements on emulsions. Corn oil was used as a lipid that remains liquid across the entire temperature range studied. The ultrasonic velocity of the buffer solution decreased with decreasing temperature and followed a similar trend as that of pure water in this temperature range.40 Conversely, the ultrasonic velocity of corn oil increased with decreasing temperature, which is similar to the ultrasonic behavior of other liquid oils in this temperature (37) Coupland, J. N. Curr. Opin. Colloid Interface Sci. 2002, 7, 445–450. (38) Unruh, T.; Bunjes, H.; Westesen, K.; Koch, M. H. J. Colloid Polym. Sci. 2001, 279, 398–403. (39) Povey, M. J. W.; Hindle, S. A.; Aarflot, A.; Hoiland, H. Cryst. Growth Des. 2006, 6, 297–301. (40) Delgross, Va.; Mader, C. W. J. Acoust. Soc. Am. 1972, 52, 1442.
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Figure 4. Temperature-dependence of the ultrasonic velocity of pure corn oil and pure buffer solution during cooling and heating at a constant rate of 0.3 °C min-1.
range.41,42 The ultrasonic velocities of the corn oil and buffer were similar (∼1475 ms-1) at a temperature of ∼19 °C. The temperature-dependence of the ultrasonic velocity of tripalmitin-in-water suspensions during heating and cooling was compared with that of pure buffer solution (Figure 5). Initially, the emulsions were heated above the melting temperature of tripalmitin, so that the droplets were completely liquid. The ultrasonic velocity of the tripalmitin emulsions increased slightly when they were cooled from 75 to 55 °C but then decreased when they cooled further from 55 to 26 °C. The ultrasonic velocity of the emulsion was much less than that of the buffer solution at these high temperatures because the ultrasonic velocity of pure oil is less than that of pure water or buffer (Figure 4). The difference in the ultrasonic velocity of the emulsion and the buffer solution became progressively smaller as the temperature was decreased because the ultrasonic velocity of pure liquid oil tends to decrease with temperature, whereas that of pure buffer tends to increase with temperature, with a crossover point at around 19 °C (Figure 4). When the emulsion was cooled below about 26 °C, there was a steep increase in the ultrasonic velocity, which can be attributed to crystallization of the lipid droplets since the ultrasonic velocity of solid fat is greater than that of liquid oil.19,20,22,26 When the emulsion was cooled further, there was a progressive decrease in ultrasonic velocity with decreasing temperature, which can be attributed primarily to the fact that the ultrasonic velocity of water (the major component in the emulsions) decreases with decreasing temperature. At the end of the cooling period (5 °C), the lipid droplets within the emulsions were completely solid. The ultrasonic velocity of the emulsions increased when the emulsions were heated from 5 to 43 °C, with values similar to those observed in the emulsions containing fully crystalline fat during cooling from 25 to 5 °C (Figure 5). The close correspondence between the ultrasonic velocities measuring during cooling and heating in the temperature range from 5 to 25 °C can be attributed to the fact that the droplets are fully crystalline. The difference in the ultrasonic velocities during cooling and heating between 25 and 43 °C can be attributed to supercooling effects as discussed earlier; that is, the lipid droplets were liquid during cooling but solid during heating in this temperature range. (41) Chanamai, R.; Coupland, J. N.; McClements, D. J. Colloids Surf., A 1998, 139, 241–250. (42) Coupland, J. N.; McClements, D. J. J. Am. Oil Chem. Soc. 1997, 74, 1559–1564.
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Figure 5. Temperature-dependence of the ultrasonic velocity of emulsified (a) 100% tripalmitin and (b) 75% tripalmitin/25% fish oil, and pure buffer solution during cooling and heating at a constant rate of 0.3 °C min-1.
There was a steep and irregular decrease in ultrasonic velocity between 44 and 62 °C, which can be attributed to the melting of crystalline tripalmitin. When the emulsions were heated from 62 to 75 °C, there was a slight decrease in the ultrasonic velocity with increasing temperature, which was similar to that observed in the emulsions containing completely liquid droplets. Plots of the ultrasonic velocity gradient (δ∆u/δT) versus temperature are a useful means of highlighting thermal transitions in materials.43,44 We therefore plotted the temperaturedependence of δ∆u/δT for the tripalmitin oil-in-water emulsions during cooling (Figure 6a) and heating (Figure 6b). These plots clearly show that there are minima in the δ∆u/δT values at particular temperatures (TmU), which correspond closely to the transition temperatures (Tm) determined by DSC. For example, a small minimum (TmU ) 29.5 °C) and a large minimum (TmU ) 25.5 °C) were observed in the δ∆u/δT values for emulsions containing pure tripalmitin during cooling (Figure 6a), which corresponded closely to the transition temperatures of the small (Tm ) 31.5 °C) and large (Tm ) 24.5 °C) peaks, respectively, observed in the DSC curves (Figure 3a). Similarly, during heating of the tripalmitin emulsions containing solid particles, a complex series of minima were observed in the δ∆u/δT curves from 42 to 63 °C (Figure 6b), which followed a pattern similar to those observed by DSC (Figure 3b). (43) Corredig, M.; Verespej, E.; Dalgleish, D. G. J. Agric. Food Chem. 2004, 52, 4465–4471. (44) Wang, Q.; Tolkach, A.; Kulozik, U. J. Agric. Food Chem. 2006, 54, 6501–6506.
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Figure 6. Plots of the ultrasonic velocity gradient (δ∆u/δT) versus temperature for 100% tripalmitin (top) and 75% tripalmitin/25% fish oil (bottom) in oil-in-water emulsions during cooling (a) and heating (b) at 0.3 °C min-1.
A close correspondence between the ultrasonic and DSC data was also found for the emulsions containing 75% tripalmitin and 25% fish oil, which were chosen for study because they exhibited thermal behavior different from that of the pure tripalmitin emulsions. During cooling, a single minimum (TmU ) 22.5 °C) was observed in the δ∆u/δT values (Figure 6a), which corresponded closely to the transition temperature (TmU ) 21.3 °C) observed in DSC (Figure 3a). During heating of the tripalmitin/ fish oil emulsions containing solidified tripalmitin, a small minimum was observed around 41.7 °C and a deep minimum was observed around 59.0 °C in the δ∆u/δT curves (Figure 5b), which corresponded closely to the values of 41.4 and 58.3 °C observed in DSC (Figure 3b). Previous studies have shown that ultrasonic measurements are sensitive to solid-liquid phase transitions in bulk fats,23,45-47 in emulsified fats,19-25 and in liposomes.48,49 To our knowledge, the present study is the first to show that temperature scanning ultrasonic velocity measurements can also be used to follow the (45) Miles, C. A.; Fursey, G. A. J.; Jones, R. C. D. J. Sci. Food Agric. 1985, 36, 215–228. (46) Maleky, F.; Campos, R.; Marangoni, A. G. J. Am. Oil Chem. Soc. 2007, 84, 331–338. (47) Singh, A. P.; McClements, D. J.; Marangoni, A. G. Food Res. Int. 2004, 37, 545–555. (48) Taylor, T. M.; Davidson, P. M.; Bruce, B. D.; Weiss, J. J. Agric. Food Chem. 2005, 53, 8722–8728. (49) Schultz, Z. D.; Levin, I. W. Biophys. J. 2008, 94, 3104–3114.
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complex thermal transitions that occur when solid lipid nanoparticles melt.
Conclusions This study has shown that temperature scanning ultrasonic velocimetry can be used to follow complex thermal transitions in tripalmitin nanoparticles. We have also shown that there is a close correspondence between the thermal transitions observed in δ∆u/δT versus temperature curves determined by ultrasound and heat flow versus temperature curves determined by DSC. Ultrasonic velocimetry is based on changes in the density and adiabatic compressibility of materials when they undergo a phase transition, whereas DSC is based on the heat absorbed or released during a phase transition. Similar to DSC, ultrasonic velocimetry is a nondestructive technique that can be used to analyze optically opaque samples without the need for any sample preparation. In addition, there are now commercial instruments available that
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can be used to carry out highly precise ultrasonic velocity measurements as a function of temperature. Temperature scanning ultrasonic velocity measurements may therefore prove to be a useful alternative to conventional DSC techniques for monitoring phase transitions in lipid systems. The main limitations of the ultrasonic technique at present are as follows: (i) measurements must be carried out at relatively slow scanning speeds (
