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Interaction between Pluronic F127 and Dioctadecyldimethylammonium Bromide (DODAB) Vesicles Studied by Differential Scanning Calorimetry Eloi Feitosa*,† and Franc-oise M. Winnik‡ †
Physics Department, IBILCE/UNESP, S~ ao Jos e do Rio Preto - SP, Brazil, and ‡Department of Chemistry and Faculty of Pharmacy, Universit e de Montr eal, Montr eal, Qu ebec, Canada Received February 16, 2010. Revised Manuscript Received October 13, 2010
A number of fundamental studies on the interactions between lipid bilayers and (ethylene oxide)-b-(propylene oxide)b-(ethylene oxide) copolymers (PEO-PPO-PEO, Pluronics) have been carried out recently as model systems for the complex behavior of cell membranes with this class of polymers often employed in pharmaceutical formulations. We report here a study by differential scanning calorimetry (DSC) of the interactions in water between Pluronic F127 (F127), and the cationic vesicles of di-n-octadecyldimethylammonium bromide (DODAB), as a function of concentration of the two components (DODAB 0.1 and 1.0 mM; F127 0.1 to 5.0 mM) and of the sample preparation protocol. The DSC studies follow the critical micellization temperature (cmt ≈ 27 °C at 1.0 mM) of F127 and the gel-liquid crystal transition (Tm ≈ 45 °C) of the DODAB bilayer and of F127/DODAB mixtures. Upon heating past Tm, vesicle/polymer mixtures undergo an irreversible conversion into mixed DODAB/F127 micelles and/or F127-bearing vesicles, depending on the relative amount of each component, together with, in some cases, residual intact F127 micelles or DODAB vesicles. Sample preparation protocol is shown to have little impact on the composition of mixed systems once they are heated above Tm.
Introduction The family of nonionic triblock copolymers composed of a hydrophobic poly(propylene oxide) central block linked to hydrophilic poly(ethylene oxide) end blocks, (EO)n(PO)m(EO)n, known under their commercial names as Pluronics or Poloxamers, have gained much interest recently in the biomedical field for their use as drug or gene delivery vehicles or sensitizers for drugresistant cells to improve drug transport or to repair biological membranes damaged by thermal burns or intense ionizing radiation exposure.1-25 Some reports, however, indicate that Pluronics can also induce bilayer permeabilization, which raises issues related to their long-term effects and their optimal dosing.24,25 To account *Address for correspondence: Department of Physics, IBILCE/UNESP Rua Cristovao Colombo, 2265 Sao Jose do Rio Preto, SP - Brazil CEP, 15054000. Phone: þ55 17 3221 22 40. Fax: þ55 17 3221 22 47. E-mail: eloi@ibilce. unesp.br.
(1) Mortensen, K. Colloids Surf., A 2001, 183, 277–292. (2) Alexandridis, P.; Holzwarth, J.; Hatton, T. Macromolecules 1994, 27, 2414– 2425. (3) Alexandridis, P.; Hatton, T. A. Colloids Surf., A 1995, 96, 1–46. (4) Hecht, E.; Hoffmann, H. Colloids Surf., A 1995, 96, 181–197. (5) Bohorquez, M.; Koch, C.; Trygstad, T.; Pandit, N. J. Colloid Interface Sci. 1999, 216, 34–40. (6) Chaibundit, C.; Ricardo, N. M.; Costa, F.; de, M.; Yeates, S. G.; Booth, C. Langmuir 2007, 28, 9229–9236. (7) Pandit, N.; Trygstad, T.; Croy, S.; Bohorquez, M.; Koch, C. J. Colloid Interface Sci. 2000, 222, 213–220. (8) Gaucher, G.; Dufresne, M.-H.; Sant, V. P.; Kang, N.; Maysinger, D.; Leroux, J.-C. J. Controlled Release 2005, 109, 169–188. (9) Almgren, M.; Stam, J. V.; Lindblad, C.; Li, P.; Stilbs, S. P.; Bahadur, P. J. Phys. Chem. 1991, 95, 5677–5684. (10) Lavasanifar, A.; Samuel, J.; Kwon, G. S. Adv. Drug Delivery Rev. 2002, 54, 169–190. (11) Kabanov, A.; Batrakova, E. V.; Alakhov, V. Y. J. Controlled Release 2002, 82, 189–212. (12) Moore, T.; Croy, S.; Mallapragada, S.; Pandit, N. J. Controlled Release 2000, 67, 191–202. (13) Pandit, N. K.; Kisaka, J. Int. J. Pharm. 1996, 145, 129–36. (14) Wenzel, J. G. W.; Balaji, K. S. S.; Koushik, K.; Navarre, C.; Duran, S. H.; Rahe, C. H.; Kompella, U. B. J. Controlled Release 2002, 85, 51–59. (15) Wanka, G.; Hoffmann, H.; Ulbricht, W. Macromolecules 1994, 27, 4145– 4159.
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for such opposite effects, researchers have undertaken systematic studies of the interactions between Pluronics and lipid vesicles, the simplest models of the cell membrane.1-4 Most data available to date stem from systems consisting of phospholipid vesicles supplemented with low concentrations of Pluronics.23-25 The fate of vesicles in concentrated Pluronic fluids has been largely ignored, although it is well-known, within the polymer scientists community, that aqueous Pluronic solutions exhibit a very rich, temperaturedependent, phase diagram.1 In dilute cold aqueous solutions, Pluronics exist as isolated chains. When the concentration or temperature exceeds critical values, the critical micellar concentration (cmc), or critical micellar temperature (cmt), respectively, chain association takes place yielding multichain polymeric micelles with a hydrophobic PPO core and a hydrated PEO corona.1-4 Under conditions of higher concentrations and/or temperatures, the polymeric micelles self-assemble into clusters, and eventually, gelation takes place.14-20 Vesicles formed by natural phospholipids are the models of choice to unravel the mechanism of the interaction of biological membranes with Pluronics, yet in the context of surface and colloid science, systems consisting of polymers and synthetic vesicles are of considerable importance, since they may form novel phases of interest from the scientific and industrial points of view or as new temperature-responsive formulations suitable for as yet unexplored applications. A number of bitail cationic surfactants, such as di-n-octadecyldimethyl ammonium bromide (16) Rassing, J.; Attwood, D. Int. J. Pharm. 1983, 13, 47–55. (17) Zhang, Y.; Lam, Y. M.; Tan, W. S. J. Colloid Interface Sci. 2005, 285, 74–79. (18) Hecht, E.; Mortensen, K.; Gradzielski, M.; Hoffmann, H. J. Phys. Chem. 1995, 99, 4866–4874. (19) Su, Y.-L.; Wei, X.-F.; Liu, H.-Z. J. Colloid Interface Sci. 2003, 264, 526–531. (20) Bakshi, M. S.; Sachar, S. Colloids Surf., A 2006, 276, 146–154. (21) Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J. J. Controlled Release 2006, 114, 100–109. (22) Liang, C.-H.; Chou, T.-H. Chem. Phys. Lipids 2009, 158, 81–90. (23) Chieng, Y. Y.; Chen, S. B. J. Phys. Chem. B 2009, 113, 14934–14942. (24) Wu, G.; Lee, K. Y. C. J. Phys. Chem. B 2009, 113, 15522–15531. (25) Chandaroy, P.; Sen, A.; Hui, S. W. J. Controlled Release 2001, 76, 27–37.
Published on Web 11/04/2010
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(DODAB), spontaneously form large unilamellar vesicles in water, whose properties can be changed by sonication, extrusion, or additives.26-40 In the case of DODAB, for which the complete phase diagram in water has been described,41,42 large to giant unilamellar vesicles form spontaneously over a wide concentration range.26-30 They have a broad size distribution,27 but exhibit well-defined gel-liquid crystal transitions around 45 and 40 °C upon heating and cooling, respectively.28,33 In aqueous solutions of concentrations higher than ∼1.0 mM, DODAB forms more complex lamellar bilayer structures, as reflected, for instance, by complex DSC thermograms and viewed by cryogenic transmission electron microscopy.42 Given the well-characterized selfassembly and thermal properties of DODAB vesicles, they are excellent probes to investigate the interactions of nonphospholipid vesicles and Pluronics in water. In selecting the Pluronic component, we sought out a polymer that has been used in previous mechanistic studies of phospholipid liposome/Pluronic systems, has a well-defined phase diagram in water, and is relevant in terms of practical applications. We chose Pluronic F127 of average molecular weight 12 600 g/mol, whose temperature/concentration phase diagram in water has been studied in detail.1,12,13,23-25 It is used in various formulations either in the gel state or as micellar solutions, some of which are past the stage of clinical trial II evaluation.1 In addition, Pluronic F127 was shown to hypersensitize multidrug-resistant tumor cells, which suggests that the polymer interacts with the membrane of tumor cells to facilitate drug internalization and justifies basic studies on model systems.1 We demonstrate in this report that high-sensitivity differential scanning calorimetry (DSC) is a simple, yet powerful tool to examine interactions between well-defined Pluronics, here F127, and vesicles, here DODAB, as a function of key parameters, such as the relative Pluronic to surfactant concentration, the temperature, and the mixing protocol. The study generates guidelines for the design of DODAB-F127-based complex fluids of desirable macroscopic properties. It provides data, which can be compared to results of studies of phospholipid liposomes/Pluronics and helps in understanding the complex interactions between Pluronics and cell membranes.
Experimental Section Materials. DODAB (Sigma-Aldrich, MW: 631.0 g/mol) was purified by recrystallization from methanol/acetone (1:3 v/v) as (26) Feitosa, E.; Alves, F. R.; Castanheira, E. M. S.; Real Oliveira, M. E. C. D. Colloid Polym. Sci. 2009, 287, 591–599. (27) Lopes, A.; Edwards, K.; Feitosa, E. J. Colloid Interface Sci. 2008, 322, 582–588. (28) Benatti, C. R.; Feitosa, E.; Fernandez, R. M.; Lamy-Freund, M. T. Chem. Phys. Lipids 2001, 111, 93–104. (29) Feitosa, E.; Barreleiro, P. C. A.; Olofsson, G. Chem. Phys. Lipids 2000, 105, 201–213. (30) Feitosa, E.; Karlsson, G.; Edwards, K. Chem. Phys. Lipids 2006, 140, 66–74. (31) Cuccovia, I. M.; Chaimovich, H.; Lissi, E.; Abuin, E. Langmuir 1997, 6, 1601–1604. (32) Segota, S.; Heimer, S.; Tezak, ^. Colloids Surf. A: Physicochem. Eng. Aspects 2006, 274, 91–99. (33) Cocquyt, J.; Olsson, U.; Olofsson, G.; Van der Meeren, P. Langmuir 2004, 20, 3906–3912. (34) Feitosa, E.; Brown, W. Langmuir 1997, 13, 4810–4816. (35) Coppola, L.; Youssry, M.; Nicotera, I.; Gentile, L. J. Colloid Interface Sci. 2009, 338, 550–557. (36) Kawamuro, M. K.; Chaimovich, H.; Abuin, E. B.; Lissi, E. A.; Cuccovia, I. M. J. Phys. Chem. 1991, 95, 1458–1463. (37) Brito, R. O.; Marques, E. F. Chem. Phys. Lipids 2005, 137, 18–28. (38) Feitosa, E.; Alves, F. R. Chem. Phys. Lipids 2008, 156, 13–16. (39) Nascimento, D. B.; Rapuano, R.; Lessa, M. M.; Carmona-Ribeiro, A. M. Langmuir 1998, 14, 7387–7391. (40) Feitosa, E.; Barreleiro, P. C. A. Prog. Colloid Polym. Sci. 2004, 128, 163–168. (41) Schulz, P. C.; Rodriguez, J. L.; Soltero-Martinez, F. A.; Puig, J. E.; Proverbio, Z. E. J. Therm. Anal. 1998, 51, 49–62. (42) Kodama, M.; Kunitake, T.; Seki, S. J. Phys. Chem. 1990, 94, 1550–1554.
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reported previously.36 Pluronic F127 (Sigma-Aldrich) was used as received. It has an average molecular weight of 12 600 g/mol, a hydrophilic/lipophilic balance (HLB) of 22, and a PEO/PPO weight ratio of 2:1. Water was deionized using a Milli-Q Plus system (Millipore).
Preparation of F127 Solutions and of DODAB Vesicle Dispersions. F127 solutions ranging in concentration from 0.1 to
10.0 mM were prepared at room temperature (22 °C) by adding water (or a preformed 1.0 mM DODAB dispersion; see below) to a weighed amount of F127. The mixture was gently stirred at room temperature until it became homogeneous. To prepare DODAB vesicles, water (or a preformed 1.0 mM F127 solution, see below) was added to a weighed amount of DODAB. The mixtures were warmed to 60 °C (i.e., above the DODAB Tm ≈ 45 °C), kept at this temperature while stirring gently for a few minutes using a magnetic stirrer, and cooled to room temperature (25 °C), as reported.30 All samples were kept at room temperature for at least 24 h prior to the DSC measurements. Dilute (0.1 mM or less) dispersions of DODAB, obtained from dilution of the 1.0 mM samples, were clear with the bluish tinge characteristic of vesicle dispersions. The DODAB critical vesicle concentration is very small (,0.01 mM),26,34 and samples of [DODAB] > 1.0 mM are turbid, most probably due to formation of more complex bilayer structures.40 Preparation of Mixed DODAB-F127 Dispersions. Four different methods (A to D) were employed to prepare the DODAB/ F127 mixtures. (1) Type A: a preformed DODAB (1.0 mM) dispersion was added at room temperature to a weighed amount of F127 to obtain a mixed system containing 10.0 mM F127. Samples of lower F127 concentrations were prepared by diluting this stock solution with the 1.0 mM DODAB dispersion. (2) Type B: a preformed F127 solution 1.0 mM heated to 60 °C was added to weighed amounts of DODAB. (3) Type C: preformed 1.0 mM DODAB dispersion and F127 solution were mixed either at 25 or at 60 °C. (4) Type D: water was added to weighed amounts of DODAB and F127; the mixture was heated to 60 °C, gently stirred magnetically at this temperature for a few minutes, and cooled to room temperature. DSC Measurements. DSC measurements were performed on a VP-DSC microcalorimeter (MicroCal Inc.) at an external pressure of ca. 180 kPa. The cell volume was 0.517 mL. Samples were degassed at 25 °C for 20 min. At least two consecutive DSC heating/cooling scans from 5 to 55 °C were performed, and unless otherwise stated, the scan rate was 20 °C/h and the prescan time was 15 min. This scan rate was selected for convenience, after confirming by using several single-component solutions and mixed systems, that changing the heating rate from 10 to 90 °C/h did not affect the DSC profile. Data were corrected for instrumental response time to take into account the effect of scan rate on the data collected. For each sample, the excess heat capacity curve was constructed by subtraction of a water vs water scan from the sample vs water scan. The data were fitted and analyzed using the software (Origin 7.0) supplied by the manufacturer. It gave the peak maximum temperature, the peak width, and the area of the transition.
Results and Discussion DSC Studies of Aqueous Pluronic F127 Solutions and of DODAB Vesicular Suspensions Prior to Mixing. Thermograms recorded upon heating F127 aqueous solutions present a single, broad endotherm (ΔT1/2 ≈ 5 °C) corresponding to the temperature-induced association of the polymers. Consecutive heating scans had identical profiles even when different scan rates were used (data not shown), and cooling scans featured a single exothermic peak centered at the same temperature as the endotherm obtained upon heating, i.e., the endo- and the exotherm peaks are symmetric. The absence of thermal hysteresis is indicative of the thermal reversibility of the temperature-dependent DOI: 10.1021/la102603a
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Figure 1. DSC traces recorded during the second heating scans of neat F127 solutions at concentrations 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mM (curves 1-7, respectively). We used three different colors for better viewing. Data collected after a prescan time 15 min at 5 or 60 °C.
Figure 2. Changes of cmt as a function of the F127 concentration in the absence and presence of DODAB 1.0 mM, obtained, respectively, from the cmt peak positions in Figures 1 and 3a.
micellization/demicellization phenomena. We recorded thermograms of F127 solutions ranging in concentration from 0.1 to 5.0 mM (Figure 1). This concentration window includes solutions below the cmc of the polymer at 20 °C (0.1-0.6 mM), around the cmc, and above it. The cmt of the solutions, taken as the temperature corresponding to the endotherm maximum, exhibits a marked concentration dependence as depicted in Figure 2. It decreases rapidly (nonlinearly) from 32.4 to 25.4 °C with increasing concentration from 0.05 to 2.0 mM. For more concentrated solutions, the decrease of cmt is less pronounced. Our observations are in general agreement with previous studies of aqueous F127 solutions, although direct comparisons are difficult, given disparities in techniques and concentrations.4,15-17,19,23,25 A thermogram of an aqueous vesicular DODAB dispersion (1.0 mM), recorded upon heating, is included in Figure 3 (top scan). It consists of a main endotherm centered at Tm = 45.6 °C and three secondary peaks, which are always observed in the heating profiles, but not during cooling scans. The origin of the secondary transitions is poorly understood.26,29,33,38,40 The main 17854 DOI: 10.1021/la102603a
Figure 3. DSC traces recorded for type A samples during the first (a) and second (b) heating scans at constant 1.0 mM DODAB and varying F127 concentration: 0.0, 0.1, 1.0, 2.0, 5.0, and 10.0 mM (curves 1-6, respectively). (c) DSC traces for type A samples consisting of 1.0 mM DODAB mixed with 0.5 mM F127, in the heating (solid curves 1 and 3) and cooling (dashed curves 2 and 4) modes. The prescan time was 15 min prior to every scan. Langmuir 2010, 26(23), 17852–17857
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Figure 4. Changes of melting temperature as a function of the F127 concentration in the presence of DODAB 1.0 mM, obtained from the melting peak position in the first and second scans (from Figure 3a and b).
endotherm is sharp (ΔT1/2 ≈ 1 °C) and corresponds to the gelliquid crystal transition.26,29 Upon cooling, it shifts to a lower temperature by ca. 5 °C. The hysteresis of the gel-liquid crystal transitions of DODAB bilayers was reported before.28,33 Mixed Pluronic F127/DODAB Samples Obtained by Dissolution at Room Temperature of F127 in a Preformed 1.0 mM DODAB Vesicular Dispersion (Method A). Mere dissolution of F127 in a dispersion of DODAB vesicles in the gel phase (room temperature) is expected to occur without diffusion of the polymer within the aqueous core of the vesicles. Hence, type A sample preparation allows one to probe the interactions of the polymer with the outer vesicle/water interface. Thermograms recorded during the first heating scan of type A DODAB/F127 systems, either as isolated chains ([F127] < cmc at room temperature, e.g., 0.1 mM) or in the form of micelles, are presented in Figure 3a. The thermograms display a transition centered at a temperature identical to the cmt of a F127 solution of equal concentration in the absence of DODAB (Figure 2, red squares). The correspondence of the cmt values implies that, prior to heating, the polymer chains remain intact in solution in the presence of vesicles. In addition to the endotherm at cmt, profiles recorded during the first heating scan of mixed systems (Figure 3a) present a second transition centered in the vicinity of the gel-liquid crystal transition of neat DODAB vesicles. Its temperature is not exactly identical to Tm (45.6 °C) measured for DODAB dispersions (Figure 3a, curve 1). To emphasize this point, we use T 0 m to identify the maximum of the gel-liquid crystal transition of the DODAB bilayer in the presence of F127. The T 0 m values increase markedly with increasing F127 concentration, as depicted in Figure 4 (black squares). The shift of T 0 m toward higher temperatures, compared to Tm, is most pronounced in the F127 premicellar concentration range. The fact that the presence of F127 affects Tm is indicative of changes in the packing density of the surfactant bilayer, which strongly suggests that some polymer chains are adsorbed on the vesicle/water interface. Figure 3b presents thermograms recorded during the second heating of the same samples. They are not identical to the profiles recorded in the first scan (Figure 3a). The endotherm for the bilayer gel-liquid crystal transition is detected only in the sample of lowest F127 concentration (0.1 mM). It is centered at a temperature slightly different from T 0 m measured in the first scan (Figure 4, red circles). Thermograms of mixed systems containing Langmuir 2010, 26(23), 17852–17857
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micellar F127 (concentration g 1.0 mM) do not feature this transition, implying that the DODAB bilayer was irreversibly damaged upon heating (first scan) past Tm in the presence of micellar F127. Significant changes of the thermograms are observed also in the temperature window around the cmt of the polymer (25-30 °C). Although the transition corresponding to cmt is still observed, it is accompanied by a new transition discernible as “shoulder” on the left side of the cmt transition (Figure 3b). This additional transition is attributed to the micellization of polymer/surfactant complexes formed at a slightly lower temperature than the cmt as a result of the disruption of the vesicles in the hot mixed systems. The vesicle disintegration in the presence of polymer aggregates is unlikely to result in a homogeneous population of mixed complexes. One can expect the formation of objects ranging from polymer-rich surfactantdecorated micelles to isolated polymer chains solubilized by small DODAB bilayer fragments bound to the PPO blocks. Upon cooling, vesicles do not form; instead, polymer/DODAB complexes of various structures coexist in solution with, possibly, surfactant-free F127 monomers. Each entity will have its own thermal signature, which accounts for the complexity of the cmt-related transitions. Figure 3c presents four consecutive scans recorded for a 1.0 mM DODAB/0.5 mM F127 mixture (type A). Scans 1 and 3 were recorded upon heating, while scans 2 and 4 were obtained upon cooling. The bilayer main transition, T 0 m, in the mixed system is lower in the cooling scan by ca. 6 °C, compared to the heating scan, as in the case of neat DODAB. Furthermore, the intensity values of the cmt and cmt0 peaks are, respectively, smaller and larger in the cooling and heating scans due to the disruption of DODAB vesicles above Tm and the formation of F127/DODAB complexes, resulting in more intense melting peak in the heating than in the cooling mode. Mixed Samples Prepared by Formation of DODAB Vesicles in the Presence of F127 Micelles (1.0 mM) (Method B). In this section, we examine systems in which vesicles were formed in the presence of polymer (sample type B) and consequently could entrap F127 in their inner core. Thermograms recorded during the first heating of the samples feature a transition in the vicinity of the gel-liquid crystal transition of polymer-free DODAB vesicles (Figure 5a), confirming that vesicles were indeed obtained by this mixing protocol. This transition is centered at the temperature T 0 m > Tm (at 50.8 and 47.6 °C, respectively, in mixtures of [DODAB] 0.1 and 1.0 mM), indicating that the bilayer is decorated with adsorbed or intercalated polymer chains. Scans recorded during the first heating (Figure 5a) present, in addition to the Tm endotherm, a transition at the cmt of F127 with a shoulder on the low-temperature side for [DODAB] = 1.0 mM. The complexity of the transition around the cmt implies that a fraction of DODAB molecules binds to the polymer or its micelles during the mixing process. Thermograms recorded during the second heating scan of mixed systems with [DODAB] 0.1 and 1.0 mM (Figure 5b) present a broad transition around cmt, reminiscent of the transition observed in the second heating of samples prepared upon dissolution of F127 in the presence of vesicles. A small characteristic transition of DODAB vesicles around Tm appears only for 1.0 mM DODAB. Hence, vesicles directly prepared in the presence of polymer (method B) disintegrate completely (at 0.1 mM DODAB) or partially (at 1.0 mM DODAB) when heated past Tm to form polymer/surfactant complexes, as did samples obtained by method A. Mixed Aqueous Samples Obtained upon Mixing at 25 or 60 °C a Micellar F127 Solution (1.0 mM) and a Preformed DODAB (1.0 mM) Vesicular Dispersion (Method C). The profile of the first heating of a sample prepared at 25 °C (blue curve DOI: 10.1021/la102603a
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Figure 5. First (a) and second (b) DSC heating traces for type B samples with 1.0 mM F127 and 0.1 and 1.0 mM DODAB (curves 2 and 3, respectively). Curve 1 is for DODAB-free F127. Data collected after a prescan time 15 min at 5 °C.
in Figure 6a) has the same overall features as the first scan recorded for type A or B mixtures, although the T 0 m endotherm is more intense in samples obtained by method C, compared to methods A or B. Profiles recorded during the second heating scan exhibit a very weak endotherm at T 0 m and a strong endotherm at cmt0 in addition to the endotherm at cmt. Note that the intensity of the melting endotherm decreases to disappear after the third thermal cycle (curve 5 in Figure 6a displays no peak), even though the scan rate was increased from 10 to 90 °C/h. Since there is no dependence of the trace profile on the scan rate (data not shown), the decrease in peak intensity is due to the increasing number of thermal cycles. Figure 6b shows DSC traces recorded for samples obtained upon addition of a vesicle dispersion preheated to 60 °C to a polymer solution also at 60 °C. The trace recorded during the first heating presents an intense endotherm at cmt0 and a weak transition at T 0 m, confirming the instability of DODAB vesicles when they are brought into contact with F127 in the bilayer liquid crystalline phase. The second heating trace displays an even weaker Tm endotherm and a sharper endotherm at around cmt. Mixed Samples Obtained upon Codissolution of 1.0 mM DODAB and F127 (Method D). Thermograms recorded for samples obtained by this method (Figure 7) do not differ 17856 DOI: 10.1021/la102603a
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Figure 6. DSC traces for 1.0 mM equimolar F127 and DODAB of type C mixtures prepared at 25 °C (a) and at 60 °C (b). In (a), data collected at scan rates 10, 20, 30, and 90 °C/h (curves 2-5, respectively). The trace for neat DODAB 1.0 mM was included for comparison (curve 1). We used three different colors for better viewing of the curves. In (b), traces of four consecutive heating (solid curves 1 and 3) and cooling (dashed curves 2 and 4) scans. Data collected after a prescan time 15 min at 5 or 85 °C.
significantly from those of samples prepared by methods A-C (25 °C). Thus, this protocol also led to the formation of vesicles, presumably incorporating polymer in their aqueous pocket as well as in the outer vesicle/water interface. They are converted to polymer/ surfactant complexes upon heating past Tm during sample preparation. The fact that the transitions corresponding to cmt and Tm are rather broad is a consequence of the overlap of two endotherms of approximately the same intensity. One should note that, even though the sample has been prepared at 60 °C, it was injected into the instrument cell at 25 °C after standing at this temperature for ca. 24 h. During the cooling and standing time, the molecules were redistributed, and structural changes may have occurred in the system. The properties of the mixed F127/DODAB system reported here based on microcalorimetry data resemble those of mixed Pluronic/phospholipid liposomes reported in the literature and refs 23-25. For instance, based on isothermal titration calorimetry studies, Wu and Lee reported that Pluronics interact with phospholipid bilayers when they are in the liquid crystalline phase (above Tm), but not in the gel phase (below Tm).24 It has also been reported that the PPO block of the Pluronic copolymers can be Langmuir 2010, 26(23), 17852–17857
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polymer concentration.25 In our DODAB/F127 system, the onset temperature may be related to the melting temperature T 0 m of the DODAB-F127 complexes, which is larger than that of neat DODAB. Overall, our interpretation of DSC data is in good accord with the conclusions reported on the Pluronic/phospholipid systems.
Conclusions
Figure 7. First and second consecutive heating traces of 1.0 mM equimolar DODAB and F127 mixed type D sample, curves 1 and 2, respectively. Data collected after a prescan time of 15 min at 5 °C.
inserted into the vesicle bilayers, whereas the PEO blocks are present on the inner and outer bilayer/water interfaces.25 In the same communication, it was reported that the onset temperature of release of F127 incorporated into the aqueous core of dioleoylphosphatidylcholine (DOPC) vesicles increases with increasing
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According to the DSC data, the type of DODAB-F127 complexes formed depends not only on the temperature and the relative amount of each component, but also on whether or not the system has been preheated above Tm prior to analysis. Prior to heating, mixtures prepared below cmt (or cmt0 ) contain primarily free F127 copolymers, complexes of F127 copolymer with bound DODAB molecules, and DODAB vesicles decorated with F127 copolymers. In addition, they may contain small amounts of virgin DODAB vesicles. Samples heated above cmt contain mixed F127/DODAB micelles, DODAB vesicles decorated with F127, and possibly surfactant-free F127 micelles. The relative amounts of the three complex species depend on the relative DODAB and F127 concentration and (at equilibrium, after some thermal cycles) to a lesser extent on the sample preparation protocol. Acknowledgment. FAPESP (Proc. 2009/00283-7) is acknowledged for supporting E.F. visit to Prof. F. Winnik’s laboratory.
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