pubs.acs.org/Langmuir © 2009 American Chemical Society
Nonsynchronous Change in the Head and Tail of Dioctadecyldimethylammonium Bromide Molecules during the Liquid Crystalline to Coagel Phase Transformation Process Fu-Gen Wu, Nan-Nan Wang, and Zhi-Wu Yu* Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China Received June 3, 2009. Revised Manuscript Received June 28, 2009 Dioctadecyldimethylammonium bromide (DODAB) is known to self-assemble into several lamellar structures in water, existing as either liquid crystalline, gel, or coagel phases. In this work, by using differential scanning calorimetry, Fourier transform infrared spectroscopy, and X-ray diffraction techniques, we have characterized the details of the phase transition mechanisms of the DODAB aqueous dispersions. It was found that the liquid crystalline converts to the coagel phase via a two-step mechanism: first to the gel phase upon cooling and then to the stable coagel phase. Although significant conformational changes in the hydrocarbon tails were observed in both steps, changes in the headgroups of DODAB were only detected in the second step. More interestingly, we found that the lipid tails change prior to the headgroups during the overall liquid crystalline to coagel phase transformation process. This is regarded as a nonsynchronicity phenomenon, which reflects the regional (head/tail) imbalance in molecular interactions. Such a nonsynchronicity phenomenon in the self-assembled aggregates composed of the medium-sized DODAB molecules will shed light on our understanding of the polymorphism and reversibility of amphiphiles including both surfactants and biomembrane phospholipids.
1. Introduction Amphiphiles are a class of molecules with hydrophilic polar heads and hydrophobic apolar tails. When dispersed in water, they will spontaneously form organized molecular aggregates such as micelles, vesicles, and tubes.1-5 Macroscopically, the physical states of the dispersions are classified as coagel, gel, liquid crystalline, hexagonal, and cubic phases, etc.6-13 The stabilities of these phases are influenced by many physical and chemical factors including temperature, solvent content, and *To whom correspondence should be addressed. Tel: (þ86)10 6279 2492. Fax: (þ86)10 6277 1149. E-mail:
[email protected]. (1) Engberts, J. B. F. N.; Kevelam, J. Curr. Opin. Colloid Interface Sci. 1996, 1, 779–789. (2) Lichtenberg, D.; Opatowski, E.; Kozlov, M. M. Biochim. Biophys. Acta 2000, 1508, 1–19. (3) Davies, T. S.; Ketner, A. M.; Raghavan, S. R. J. Am. Chem. Soc. 2006, 128, 6669–6675. (4) Deng, M. L.; Huang, X.; Wu, R. L.; Wang, Y. L. J. Phys. Chem. B 2008, 112, 10509–10513. (5) Wang, C.; Yin, S. C.; Chen, S. L.; Xu, H. P.; Wang, Z. Q.; Zhang, X. Angew. Chem., Int. Ed. 2008, 47, 9049–9052. (6) Seddon, J. M.; Templer, R. H. In Handbook of Biological Physics; Lipowsky, R., Sackmann, E., Eds.; Elsevier: Amsterdam, 1995; Vol. 1, pp 97-160. (7) Kawai, T.; Umemura, J.; Takenaka, T.; Kodama, M.; Seki, S. J. Colloid Interface Sci. 1985, 103, 56–61. (8) Cassin, G.; De Costa, C.; Van Duynhoven, J. P. M.; Agterof, W. G. M. Langmuir 1998, 14, 5757–5763. (9) Sperline, R. P. Langmuir 1997, 13, 3715–3726. (10) Palma, S.; Manzo, R. H.; Allemandi, D.; Fratoni, L.; Lo Nostro, P. Langmuir 2002, 18, 9219–9224. (11) Wang, W.; Li, L. M.; Xi, S. Q. J. Colloid Interface Sci. 1993, 155, 369–373. (12) Cross, W. M.; Kellar, J. J.; Miller, J. D. Appl. Spectrosc. 1992, 46, 701–704. (13) Kodama, M.; Seki, S. Prog. Colloid Polym. Sci. 1983, 68, 158–162. (14) Yu, Z. W.; Quinn, P. J. Mol. Membr. Biol. 1998, 15, 59–68. (15) Templer, R. H.; Seddon, J. M.; Warrender, N. A.; Syrykh, A.; Huang, Z.; Winter, R.; Erbes, J. J. Phys. Chem. B 1998, 102, 7251–7261. (16) Squires, A.; Templer, R. H.; Ces, O.; Gabke, A.; Woenckhaus, J.; Seddon, J. M.; Winter, R. Langmuir 2000, 16, 3578–3582. (17) Wu, F. G.; Chen, L.; Yu, Z. W. J. Phys. Chem. B 2009, 113, 869–872. (18) Gao, W. Y.; Chen, L.; Wu, R. G.; Yu, Z. W.; Quinn, P. J. J. Phys. Chem. B 2008, 112, 8375–8382.
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cosolute concentration. Because amphiphile dispersions can be used as biomembrane models14-18 and have wide applications in various fields such as sensors,19 drug delivery systems,20 and microreactors,21 tremendous efforts have been made to understand the basic principles governing their phase stabilities and phase transition mechanisms. Although the phase behaviors of amphiphiles (especially the lipid systems) have been widely studied from various aspects, an in-depth understanding of the polymorphism and reversibility of the phase transitions of amphiphiles is still lacking. A longstanding question is the molecular mechanisms of the occurrence of metastable phases. To address this question, we propose that the “headgroups” (the polar region) and “tails” (the apolar region) of some amphiphiles may change nonsynchronously in response to changes of temperature or other conditions. Knowledge of such nonsynchronicity may provide new insight into this problem. It has been noticed that the nonsynchronicity phenomena have been reported in the study of protein folding/unfolding processes and polymer phase transitions. When a protein undergoes folding/unfolding processes, one domain may change prior to other domains.22-24 In the polymer phase transition process, the side chains may change earlier than the main chain.25,26 (19) Savariar, E. N.; Ghosh, S.; Gonzalez, D. C.; Thayumanavan, S. J. Am. Chem. Soc. 2008, 130, 5416–5417. (20) Samad, A.; Sultana, Y.; Aqil, M. Curr. Drug Delivery 2007, 4, 297–305. (21) Michel, M.; Winterhalter, M.; Darbois, L.; Hemmerle, J.; Voegel, J. C.; Schaaf, P.; Ball, V. Langmuir 2004, 20, 6127–6133. (22) Zhou, P.; Xie, X.; Knight, D. P.; Zong, X. H.; Deng, F.; Yao, W. H. Biochemistry 2004, 43, 11302–11311. (23) Shashilov, V. A.; Lednev, I. K. J. Am. Chem. Soc. 2008, 130, 309–317. (24) Ashton, L.; Barron, L. D.; Czarnik-Matusewicz, B.; Hecht, L.; Hyde, J.; Blanch, E. W. Mol. Phys. 2006, 104, 1429–1445. (25) Sun, B. J; Lin, Y. N; Wu, P. Y. Appl. Spectrosc. 2007, 61, 765–771. (26) Curtis, M. D.; Nanos, J. I.; Moon, H.; Jahng, W. S. J. Am. Chem. Soc. 2007, 129, 15072–15084.
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Figure 1. Molecular structure of DODAB.
These nonsynchronicity phenomena are very important for the maintenance/regulation of the properties and functions of proteins and polymers. Recent experimental,27 theoretical,28 and simulation29 works have shown that the formation of a crystalline phase in polymers is preceded by precursor structures with polymer backbone rigidity different to that of the initial amorphous phase, also giving good examples of the nonsynchronous change of the side chain and backbone of polymers. Thus, here comes a question that whether the nonsynchronous local structural changes can also occur in the phase transitions of amphiphiles. Up to now, however, no explicit studies have been devoted to answer this question. In this contribution, a simple structured lipid molecule, dioctadecyldimethylammonium bromide (DODAB), was selected to address the nonsynchronicity question. DODAB is one typical example of the bilayer forming double-chained cationic surfactants that often behave similarly to biomembrane lipids in an aqueous environment.30 Its molecular structure is schematically shown in Figure 1, which can be divided into two parts: the polar head region composed of N(CH3)2þ and the apolar tail region composed of two long hydrocarbon chains. A study on the phase behavior of DODAB dispersed in water can deepen our understanding of the phase transition properties of biological membranes. Other than being used as a membrane-mimicking model molecule, DODAB has widespread applications such as gene therapy, drug solubilization, surface recognition in nanotemplates, kinetics of chemical reactions, and catalysis.31 Phase behaviors of DODAB aggregates are believed to have a close relationship to the above applications. They may also be fundamental to understand properties of various binary or more complex systems such as DODAB-phospholipid,32 DODAB-surfactant,33 DODAB-sterol,34 DODAB-DNA,35 DODAB-protein,36 DODAB-polymer,37 and DODABpolymeric particle/latex.38 The neat DODAB aqueous dispersions have been investigated from various aspects, including preparation methods,31,39-43 (27) Soccio, M.; Nogales, A.; Lotti, N.; Munari, A.; Ezquerra, T. A. Phys. Rev. Lett. 2007, 98, 037801/1–037801/4. (28) Strobl, G. Prog. Polym. Sci. 2006, 31, 398–442. (29) Gee, R. H.; Lacevic, N.; Fried, L. E. Nat. Mater. 2006, 5, 39–43. (30) Umemura, J.; Kawai, T.; Takenaka, T.; Kodama, M.; Ogawa, Y.; Seki, S. Mol. Cryst. Liq. Cryst. 1984, 112, 293–309. (31) Brito, R. O.; Marques, E. F. Chem. Phys. Lipids 2005, 137, 18–28. (32) Sobral, C. N. C.; Soto, M. A.; Carmona-Ribeiro, A. M. Chem. Phys. Lipids 2008, 152, 38–45. (33) Coppock, J. D.; Krishan, K.; Dennin, M.; Moore, B. G. Langmuir 2009, 25, 5006–5011. (34) Benatti, C. R.; Epand, R. M.; Lamy, M. T. Chem. Phys. Lipids 2007, 145, 27–36. (35) Kikuchi, I. S.; Carmona-Ribeiro, A. M. J. Phys. Chem. B 2000, 104, 2829– 2835. (36) Carvalho, L. A.; Carmona-Ribeiro, A. M. Langmuir 1998, 14, 6077–6081. (37) Klebanau, A.; Kliabanova, N.; Ortega, F.; Monroy, F.; Rubio, R. G.; Starov, V. J. Phys. Chem. B 2005, 109, 18316–18323. (38) Correia, F. M.; Petri, D. F. S.; Carmona-Ribeiro, A. M. Langmuir 2004, 20, 9535–9540. (39) Lopes, A.; Edwards, K.; Feitosa, E. J. Colloid Interface Sci. 2008, 322, 582– 588. (40) Feitosa, E.; Karlsson, G.; Edwards, K. Chem. Phys. Lipids 2006, 140, 66–74. (41) Wang, L.; Song, Y. H.; Han, X. J.; Zhang, B. L.; Wang, E. K. Chem. Phys. Lipids 2003, 123, 177–185. (42) Feitosa, E.; Barreleiro, P. C. A.; Olofsson, G. Chem. Phys. Lipids 2000, 105, 201–213. (43) Feitosa, E.; Brown, W. Langmuir 1997, 13, 4810–4816.
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phase behaviors,31,44-47 and structural characterizations of vesicles or other aggregation forms.48-50 In particular, thermotropic phase behaviors of the system have been carefully examined. Coagel, gel, and liquid crystalline phases have been observed.44 Among them, the coagel phase is said to be the “poorly hydrated multilamellar polycrystalline suspension”.9,51 A similar system, the dioctadecyldimethylammonium chloride (DODAC)-water system, has also been studied from both calorimetric and spectroscopic aspects,30,52-54 which provides complementary and comparative information for the DODAB-water system. To better understand the reversibility of the phase transition between the coagel and the liquid crystalline phases of DODAB, we have developed a strategy to examine individual functional groups of the molecule during the phase transformation. We are curious to know if the head and tail groups of DODAB take identical pace in response to temperature changes. In this work, by using differential scanning calorimetry (DSC), time-resolved Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction (XRD) techniques, we have characterized the details of the coagel to liquid crystalline, liquid crystalline to gel, and gel to coagel phase transformations of DODAB molecules. It is found that, during the conversion of the liquid crystalline to the coagel phase through the metastable gel phase, the organization of the head and tail portions undergoes changes nonsynchronously. It should be noted that different from proteins and polymers, which are macromolecules, the examined lipids in this work are medium-sized molecules that self-assemble into ordered lamellar structures. The discovery of the nonsynchronicity phenomenon in such lamellar structures composed of the medium-sized lipid molecules has profound significance in understanding the nature of the phase transformation processes of amphiphiles.
2. Experimental Section 2.1. Sample Preparation. DODAB with a purity better than 99% was purchased from Acros Organics (Belgium). Double deionized water with a resistivity of 18.2 MΩ cm was used for the hydration and suspension of the DODAB samples. The lipid/ water ratio was 1/2 (w/w). Homogeneous lipid dispersion was prepared by repeated thermal cycling between 20 and 70 °C. The initial coagel phase was obtained by slowly cooling the sample at the liquid crystalline phase to 20 °C.44 2.2. DSC. Calorimetric data were obtained with a differential scanning calorimeter DSC821e equipped with the high sensitivity sensor HSS7 (Mettler-Toledo Co., Switzerland). 2.3. FTIR Spectroscopy. FTIR spectra were recorded using a Nicolet 5700 FTIR spectrometer with a DTGS detector in the range of 4000 to 900 cm-1 with a spectral resolution of 2 cm-1 and (44) Kodama, M.; Kunitake, T.; Seki, S. J. Phys. Chem. 1990, 94, 1550–1554. (45) Schulz, P. C.; Rodriguez, J. L.; Soltero-Martinez, E. A.; Puig, J. E.; Proverbio, Z. E. J. Therm. Anal. Calorim. 1998, 51, 49–62. (46) Benatti, C. R.; Tiera, M. J.; Feitosa, E.; Olofsson, G. Thermochim. Acta 1999, 328, 137–142. (47) Cocquyt, J.; Olsson, U.; Olofsson, G.; Van der Meeren, P. Colloid Polym. Sci. 2005, 283, 1376–1381. (48) Benatti, C. R.; Feitosa, E.; Fernandez, R. M.; Lamy-Freund, M. T. Chem. Phys. Lipids 2001, 111, 93–104. (49) Goncalves da Silva, A. M.; Romao, R. S.; Lucero Caro, A.; Rodriguez Patino, J. M. J. Colloid Interface Sci. 2004, 270, 417–425. (50) Jung, M.; Hubert, D. H. W.; Van Veldhoven, E.; Frederik, P. M.; Blandamer, M. J.; Briggs, B.; Visser, A. J. W. G.; Van Herk, A. M.; German, A. L. Langmuir 2000, 16, 968–979. (51) Cameron, D. G.; Umemura, J.; Wong, P. T. T.; Mantsch, H. H. Colloids Surf. 1982, 4, 131–145. (52) Kodama, M.; Kuwabara, M.; Seki, S. Thermochim. Acta 1981, 50, 81–91. (53) Laughlin, R. G.; Munyon, R. L.; Fu, Y. C.; Fehl, A. J. J. Phys. Chem. 1990, 94, 2546–2552. (54) Kawai, T.; Umemura, J.; Takenaka, T.; Kodama, M.; Ogawa, Y.; Seki, S. Langmuir 1986, 2, 739–743.
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a zero filling factor of 2. The precision of the wavenumber is better than 0.1 cm-1. Samples were coated onto the inner surfaces of a pair of CaF2 windows, which were mounted on a Linkam heating-cooling stage (Linkam Scientific Instruments, United Kingdom) for temperature control ((0.1 °C). The exact temperature of the sample between two CaF2 windows was measured by a Pt100 standard resistor connected with a digital multimeter. For each spectrum, eight scans were performed, and spectra were recorded about every 15 s. 2.4. XRD. The repeat spacings of the lamellar structures were determined with synchrotron small-angle X-ray scattering technique (SAXS). The experiments were performed at the beamline 1W2A of the Beijing Synchrotron Radiation Facility (BSRF) (λ = 1.54 A˚). A standard silver behenate sample was used for the calibration of diffraction spacings. X-ray scattering intensity patterns were recorded during 30 s of exposure of the sample to the synchrotron beam. A Linkam thermal stage (Linkam Scientific Instruments) was used for temperature control ((0.1 °C). The X-ray powder diffraction intensity data were analyzed using the program Fit2D. The packing state of the lipid hydrocarbon chains in the coagel phase of a DODAB aqueous dispersion at 20 °C was investigated by conventional powder XRD on a Bruker D8 Advance X-ray diffractometer using Cu KR radiation (λ = 1.54 A˚). The detected angles (2θ) were in the range of 10-50°, corresponding to the wide-angle X-ray diffraction (WAXS) region.
3. Results and Discussion 3.1. DSC Measurements. Thermograms of the DODAB dispersion during first heating, cooling, and immediate second heating at a scanning rate of 0.5 °C/min are shown in Figure 2.
Figure 2. DSC results of the DODAB-H2O system. The scan rate was 0.5 °C/min.
Upon heating from 20 to 70 °C, the DODAB dispersion undergoes a phase transition from its initial coagel phase to liquid crystalline phase with the onset and peak temperatures of 50.3 and 54.5 °C, respectively. Upon cooling, the liquid crystalline phase converts first to the gel phase with the onset and peak temperatures of 43.6 and 43.0 °C, respectively. These two phase transition temperatures are in good agreement with literature data.44 Upon further cooling, a new exothermic peak (broad) at the temperature range of 37 to 23 °C occurs. This is assigned to the gel to coagel transition. As the second heating produces exactly the same thermogram as the first heating, the phase state at the end temperature of the cooling process should be the coagel phase. The fact that the total enthalpy loss of the two transitions during the cooling process (102 kJ/mol) equals the enthalpy gain of the first/second heating process (104 kJ/mol) also supports these analyses. Thus, the phase transition sequences during the heating and cooling processes are different: upon heating, the initial coagel phase converts to liquid crystalline phase; while during cooling, the liquid crystalline phase first transforms into the gel phase and then converts to the final coagel phase. We should note that during cooling, the end point of the first exothermic peak (the liquid crystalline to gel transition) and the onset of the second exothermic peak (the gel to coagel transition) merged. It is thus difficult to accurately determine the structural (using SAXS) and spectroscopic (using FTIR) features of this intermediate gel phase. However, faster cooling from 70 to 20 at 5 °C/min helps us to capture this intermediate gel phase. As demonstrated in Figure 3A, cooling from 70 to 20 at 5 °C/min gave only the liquid crystal to gel transition, with the onset temperature and enthalpy value consistent with that of the first exothermic peak during cooling in Figure 2. Immediate reheating from 20 to 70 at 1 °C/min after the faster cooling produced two peaks: an exothermic peak at 23.5 °C and an endothermic peak at 54.6 °C. The endothermic peak is easily recognized as the coagel to liquid crystalline transition based on the transition temperature and enthalpy. The exothermic peak is assigned as the gel to coagel transition because the total enthalpy loss of the exothermic peak during cooling and the first exothermic peak during heating are equal to the enthalpy gain of the second endothermic peak during heating. Another way to characterize the transition is to incubate the fast-cooled sample at 20 °C and record the relaxation process. The exothermic curve is presented in Figure 3B, which shows that the gel phase relaxed to the coagel phase within 8 min. From the above analyses, we can see that the cooling rate is very important for the observed phase behavior. The slow cooling (0.5 °C/min) results in two successive phase transitions (liquid crystalline to gel and gel to coagel), while the faster cooling (5 °C/min) results in only the liquid crystalline to gel phase
Figure 3. DSC results of the DODAB-H2O system. (A) Cooling from 70 to 20 at 5 °C/min and reheating to 70 at 1 °C/min. (B) After it was cooled from 70 to 20 at 5 °C/min, the sample was incubated at 20 °C for 30 min.
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Figure 4. Partial FTIR absorbance spectra of the three phases (liquid crystalline, gel, and coagel) of the DODAB-H2O system.
transition, and the gel to coagel transition was observed during the isothermal or subsequent reheating process. The key reason for the different phase behaviors at the two cooling rates is the nuclei formation of the coagel phase from the gel medium. During slow cooling at 0.5 °C/min, the time for the nuclei formation of the coagel phase is adequate, and enough amount of nuclei of the coagel phase have formed at the end of the liquid crystalline to gel transition, while during faster cooling at 5 °C/min, the nuclei of the coagel phase cannot form readily at the given time. Because the water content in the coagel phase is much less than that in the gel phase (see the X-ray data in the later section), the required time for the nuclei formation of the coagel phase may reflect the relative difficulty of the dehydration of lipid headgroups from the initial gel phase. 3.2. FTIR Measurements. The amphiphilic DODAB molecule can be divided into two parts: the headgroup N(CH3)2þ and the hydrocarbon tail region. We selected the CH3-(Nþ) and CH2 groups as intrinsic IR probes for monitoring the changes of the head and tail regions, respectively, to see how these groups respond during various phase transitions. The partial FTIR absorbance spectra of the three phases (liquid crystalline, gel, and coagel) of DODAB-H2O system are summarized in Figure 4. The differences of the three phases can be identified and compared. We will carefully show how these three phases change during the heating and cooling processes in the following time-resolved FTIR experiments. Shown in Figure 5 are the temperature-dependent changes of the time-resolved FTIR absorbance spectra of the main probing groups of DODAB observed during the coagel to liquid crystalline transition at 0.5 °C/min. The 3000 to 2800 cm-1 region (Figure 5A) contains the CH2 asymmetric and symmetric stretching bands. The coagel to liquid crystalline transition at around 54 °C is characterized by a shift toward higher wavenumbers and an increase of the width of the bands due to the CH2 stretching vibrations. At the coagel phase (20 °C), the CH2 asymmetric and symmetric stretching bands center at around 2916.1 and 2850.5 cm-1. After the phase transformation, the respective wavenumbers of the two bands are found at 2923.0 and 2853.1 cm-1 in the liquid crystalline phase at 70 °C. The representative wavenumbers of a few vibration modes at the three concerned phases are listed in Table 1. The characteristic wavenumber shifts have been used frequently to follow the conformational order of the lipid hydrocarbon chains and the trans-gauche Langmuir 2009, 25(23), 13394–13401
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isomerization of the CH2 groups in lipid tail regions.17,55 The increase of wavenumbers is partly due to the increase in the gauche conformers of hydrocarbon chains and partly due to the change in density or packing state of the hydrocarbon chains.56 The origin of the increase in bandwidth is the augmentation of the rotational motion of the hydrocarbon chains.7 At higher temperatures in the liquid crystalline phase, the CH2 stretching bands are broader, indicating higher mobility of these groups and their disordered packing. The 1510 to 1430 cm-1 region (Figure 5B) contains mainly two absorption bands. The band centered at 1487-1490 cm-1 is ascribed to the asymmetric deformation vibration of the methyl groups attached to the Nþ atom in the hydrophilic part.7,11,54,57 It is a key band for monitoring the structural behavior of the hydrophilic part of the amphiphilic molecule, which is known to be sensitive to the extent of disorder and the packing of the headgroups.11,57 The higher wavenumber and sharper shape of the band at 1489.2 cm-1 in the coagel phase suggests the ordering and dehydration of polar headgroups. At above 54 °C, it shifts to lower wavenumber (1487.3 cm-1) and becomes broader during the transition to the liquid crystalline phase, which is attributed to a relatively disordered, well-hydrated headgroups. The other band in this region is from the CH2 scissoring vibration centered at 1471.2 cm-1 in the coagel phase and 1466.8 cm-1 in the liquid crystalline phase. This band is very sensitive to the intermolecular forces and can be served as a key band for examining the state of packing of the hydrocarbon chains in various phases.11,30 In the coagel phase, the single peak centered at 1471.2 cm-1 indicates that the hydrocarbon transzigzag planes are packed in parallel with each other (subcell structure being orthorhombic parallel or triclinic).7,30 While in the liquid crystalline phase, the broad band at 1466.8 cm-1 suggests that the hydrocarbon chains are in the fused state.30,58 The gauche conformers become more significant, and the hydrocarbon chains are more disordered packed in the liquid crystalline phase. Faster cooling from 70 to 20 at 5 °C/min results in a transformation from liquid crystalline to gel phase (Figure 6). In Figure 6A, the asymmetric and symmetric stretching bands at 2923.0 and 2853.1 cm-1 in the liquid crystalline phase at 70 °C shift to lower wavenumbers at 2917.9 and 2850.2 cm-1 in the gel phase at 20 °C, indicating the ordering of the lipid hydrocarbon chains (from gauche to trans). Meanwhile, the CH2 scissoring band at 1466.8 cm-1 (broad) in the liquid crystalline phase shifts to 1467.5 cm-1 in the gel phase (Figure 6B). A comparison of the two representative curves is illustrated in the inset of Figure 6B, showing clearly the much sharper band in the gel phase than that in the liquid crystalline phase. The band at 1467.5 cm-1 indicates that the hydrocarbon chains are packed in the hexagonal lattice.11,30 Most interestingly, as indicated by the dotted line in Figure 6B, the asymmetric headgroup methyl deformation band centered at ∼1487 cm-1 remains almost unchanged in its band position and general feature (band contour) during the liquid crystalline to gel phase transformation process, which can be seen more clearly in the two stacked spectra in the inset of Figure 6B. These results indicate that during the liquid crystalline to gel phase transformation process, the N(CH3)2þ headgroups of DODAB remain unchanged in the hydration and conformation, with only the (55) Nabet, A.; Auger, M.; Pezolet, M. Appl. Spectrosc. 2000, 54, 948–955. (56) Umemura, J.; Cameron, D. G.; Mantsch, H. H. Biochim. Biophys. Acta 1980, 602, 32–44. (57) Wong, T. C.; Wong, N. B.; Tanner, P. A. J. Colloid Interface Sci. 1997, 186, 325–331. (58) Snyder, R. G. J. Chem. Phys. 1967, 47, 1316–1360.
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Figure 5. Time-resolved FTIR absorbance spectra of DODAB during the coagel to liquid crystalline phase transition process at 0.5 °C/min (the spectra are ∼1 °C apart). (A) The 3000-2800 cm-1 region. (B) The 1510-1430 cm-1 region. Table 1. Partial FTIR Absorption Bands of the DODAB-H2O System in the Coagel, Liquid Crystalline, and Gel Phasesa coagel (20 °C)
liquid crystalline (70 °C)
gel (20 °C)
assignmentb
2916.1 2923.0 2917.9 νasCH2 2850.5 2853.1 2850.2 νsCH2 1489.2 1487.3 1487.4 δasCH3-(Nþ) 1471.2 1466.8 1467.5 δCH2, scissoring a Band wavenumbers in cm-1. b ν-stretching and δ-bending.
hydrocarbon chains in the tail region changing in the conformational order and packing state. Figure 7 shows the time-resolved FTIR spectra during the isothermal formation of the coagel phase of the lipid aggregates at 20 °C. From Figure 7A, we can see that the CH2 conformational order changes little during the gel to coagel transformation process (see the dotted line in Figure 7A), and in Figure 7B, the band at 1487.4 cm-1 (broad) in the gel phase shifts to 1489.2 cm-1 (narrower) in the coagel phase, showing the ordering and substantial dehydration of the lipid headgroups. Furthermore, the band at 1467.5 cm-1 in gel phase shifts to 1471.2 cm-1 in the coagel phase within minutes, which indicates the change in the lipid hydrocarbon chain packing state from hexagonal to orthorhombic/triclinic. So, during the cooling and subsequent isothermal processes (Figure 6 and 7), the initial liquid crystalline phase converts to the final coagel phase via a two-step mechanism: At the first step, the lipid headgroups remain unchanged as the liquid crystalline phase transforms to the gel phase, while the lipid tail regions undergo significant changes. At the second step, both the lipid headgroups and the tail regions change markedly, corresponding to dehydration of the headgroups and further rearrangements of the tail regions as the gel phase converts to the coagel phase. That is, the lipid tail regions change prior to the lipid headgroups during the overall liquid crystalline to coagel phase transformation process, which is regarded as a nonsynchronicity phenomenon. It is for the first time to the best of our knowledge that we collected evidence at the molecular/submolecular level that the hydrophilic headgroups and hydrophobic tails of lipid molecules can change nonsynchronously during phase transformations. 3.3. Nonsynchronicity in the Liquid Crystalline to Coagel Phase Transformation Process. The physical appearances of the concerned phases are depicted in Figure 8. As shown in the figure, the liquid crystalline phase at 70 °C (Figure 8A) and the gel phase at 20 °C (Figure 8B) of DODAB aqueous dispersions are 13398 DOI: 10.1021/la901989j
both (semi)transparent and filled with small bubbles. There is no visible difference in the appearances of these two phases. To recall the FTIR results that these two phases differ only in their hydrocarbon tail arrangement, we can infer that the “appearances” of the two phases are governed by the structure of the lipid headgroups, not that of the tail regions. Upon further incubation at 20 °C, white patches formed in the gel phase (Figure 8C), which indicates the formation of the coagel phase (poorly hydrated crystalline phase). Finally, at the end of the 30 min incubation, the gel phase converted completely to the coagel phase, and the medium showed a complete white appearance (Figure 8D). The main differences between the gel and the coagel phases are the hydration degree of the lipid headgroups and the packing state of the hydrocarbon tails. As the change in the lipid tail arrangement alone cannot result in the white patches, the appearance of the white patches reflects the dehydration process of the headgroups of DODAB during the gel to coagel transformation process. The results of these photographs are consistent with the conclusion that almost no change occurs in the headgroups of DODAB during the liquid crystalline to gel transformation process. To determine the lamellar repeat distances of the three phases (the liquid crystalline, gel, and coagel phases), SAXS experiments were carried out, and the results are shown in Figure 9A. As to the liquid crystalline and gel phases, the reciprocal spacings (S) of the first two (or three) diffraction orders show a ratio of 1:2 (or 1:2:3), revealing that both of the structures are lamellar. The calculated d spacings (d = 1/S) for the liquid crystalline and gel phases are 9.23 and 7.55 nm, respectively. For the coagel phase, besides the first order at S = 0.27 nm-1, some additional periodical Bragg peaks for higher orders also appear in the larger S value region (data not shown). The calculated d spacing value was 3.70 nm, which is in good agreement with the data reported by Jung and co-workers.59 The wide-angle (10-50°) scattering result of the coagel phase is shown in Figure 9B, which is characterized by a large number of sharp peaks. Such a feature clearly shows that the coagel phase is a crystalline phase. Previous work has identified DODAB 3 2H2O as the stoichiometric composition of the crystal, which coexists with an excess water phase.45,59 By combining these XRD data (SAXS and WAXS) and the previous FTIR analyses, an illustrative model showing the (59) Jung, M.; German, A. L.; Fischer, H. R. Colloid Polym. Sci. 2001, 279, 105– 113.
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Figure 6. Time-resolved FTIR absorbance spectra of DODAB during the liquid crystalline to gel phase transition process at 5 °C/min (the
spectra are ∼1.25 °C apart). (A) The 3000-2800 cm-1 region. (B) The 1510-1430 cm-1 region. The inset figure in panel B is the liquid crystalline phase at 70 °C and the gel phase at 20 °C in the 1510-1430 cm-1 region.
Figure 7. Time-resolved FTIR absorbance spectra of DODAB during the gel to coagel phase transition process at 20 °C. (A) The 3000-2800 cm-1 region. (B) The 1510-1430 cm-1 region.
different states of the three phases is given in Figure 10. As compared with the d space value (5-7 nm)60 of phosphatidylcholines (PCs) or phosphatidylethanolamines (PEs) with the same hydrocarbon chain length and much more complex structures of the polar region, the big d value (9.23 nm) of the liquid crystalline phase of DODAB indicates a “swollen” state incorporated with a large amount of interlamellar “intermediate” water. Upon cooling to the gel phase, parts of the “intermediate” water molecules are squeezed out of the bilayers and the repeat distance d decreases to 7.55 nm, with the fact in mind that the ordering of the lipid tails during the liquid crystalline to gel transition increases the d value. At this stage, there is still a large quantity of interlamellar
“intermediate” water (note that the bilayer thicknesses without the water layer of the liquid crystalline and gel phases are only 3.32 and 3.42 nm, respectively61). Recalling the FTIR results, the headgroups at the gel phase are still well hydrated as that at the liquid crystalline phase, and the loss of these “intermediate” water molecules does not influence the strongly bound water layers of the DODAB headgroups. Finally, the conversion of the gel to coagel phase results in a drastic decrease of the d value from 7.55 to 3.70 nm, accompanied by a complete loss of the interlamellar “intermediate” water layer, a partial dehydration of the lipid headgroups, and a further ordering of the hydrocarbon tails. To sum up, the XRD results show that during the liquid crystalline to gel transition, only part of the interlamellar “intermediate” water loses (with no significant change of the d value); and during the gel to coagel transition, not only the “intermediate” water but also the strongly bound water lose (with a drastic change of the d value). These results correlate well with the above analyses of the FTIR (Figures 6 and 7) and photograph (Figure 8) results. It is worth mentioning that upon heating, the coagel phase of DODAB converts to the liquid crystalline phase, while upon cooling, the liquid crystalline phase converts to the coagel phase
(60) Marsh, D. CRC Handbook of Lipid Bilayers; CRC Press: Boca Raton, 1990.
(61) Saveyn, P.; Van der Meeren, P.; Zackrisson, M.; Narayanan, T.; Olsson, U. Soft Matter 2009, 5, 1735–1742.
Figure 8. Photographs of the DODAB-H2O system. (A) The
liquid crystalline phase (∼70 °C). (B) The gel phase (20 °C, 0 min). (C) The gel and coagel phases coexisted in the same medium (20 °C, 6 min). (D) The coagel phase (20 °C, 30 min).
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Figure 9. (A) Representative SAXS data showing the lowest order Bragg peaks of the liquid crystalline, gel, and coagel phases of the DODAB aqueous dispersion. (B) The WAXS result of the coagel phase of the DODAB dispersion.
Figure 10. Model of the transformation processes of the liquid crystalline, gel, and coagel phases of the DODAB aqueous dispersion. The “intermediate” water in the interlamellar space of the liquid crystalline phase refers to the loosely bounded water, which does not affect the hydration properties of the lipid headgroups.
via the metastable gel phase. The different phase transition sequences during heating and cooling are also observed in phospholipid aggregates.62 The hidden mechanism for the discrimination of the phase transition sequences during the heating and cooling processes of lipid aggregates is largely unknown. As to the DODAB-water system, we will give a possible explanation to this problem at the content of the nonsynchronicity phenomenon. During the first stage of the heating process (at temperatures below the onset temperature of the coagel to liquid crystalline transition), the mobility of the water molecules increases with increasing temperature, while at the same time the lipid tails gain some energy and are easier to change their state to some extent. However, at this temperature range, the changes of the headgroups and tails of the lipid molecule are still not evident. When the temperature approaches the onset temperature of the coagel to liquid crystalline phase transition, the lipid hydrocarbon tails melt, accompanied with increased solvation of the polar (62) Lewis, R. N. A. H.; McElhaney, R. N. Biophys. J. 1993, 64, 1081–1096.
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headgroups of lipids. During the whole coagel to liquid crystalline phase transition process, the salvation of the lipid headgroups and the rearrangement of the lipid tails occur quite cooperatively. Upon cooling, the headgroups of the DODAB molecules at the initial liquid crystalline phase are well hydrated, the translational and flip-flop motions are quite easy, and chain conformational disorder dominates.31 When the temperature decreases, the loosely packed tails are easier to change their packing and conformations as compared with the headgroups. The resultant phase is the metastable gel phase. The different “sensitivity” of the headgroups and tails to the change of temperature during cooling reflect the relative strength of the intra- and intermolecular forces between the head and the tail regions. For DODAB molecules, the interaction forces at the head region are mainly the attractive electrostatic forces between the charged N(CH3)2þ group and the counterion Br- and the repulsing electrostatic forces between the neighboring charged N(CH3)2þ groups. No intermolecular attractions exist between the neighboring headgroups. The relative balance of these forces is almost constant within a small temperature range. The orientation change of the headgroups during cooling, therefore, is more difficult as compared with the tails, which explains our observation that the dehydration process of DODAB headgroups occurs only after substantial rearrangements of the lipid tails. The consequence is that the headgroups and tails of DODAB molecules change nonsynchronously. The time lag of the change of headgroups during cooling is the reason for the occurrence of the intermediate gel phase, which only differs in the arrangement of the lipid tails as compared with that of the liquid crystalline phase. The different phase transition sequences of DODAB during the heating and cooling processes can also be described as the synchronous change during the coagel to liquid crystalline transition upon heating and the nonsynchronous change during the liquid crystalline to coagel transition upon cooling. The formation of the gel phase upon cooling from the liquid crystalline phase is the result of the nonsynchronous change during the liquid crystalline to coagel transition, which contributes to the polymorphism of the DODAB-water system, During the liquid crystalline to coagel phase transition process of the DODAB molecules, the change of lipid tails upon cooling from the liquid crystalline phase did not result in the change of the lipid headgroups, which is quite different from the dimirystoylphosphatidylethanolamine (DMPE)-water system.62 In the latter case, the ordering of the DMPE tails upon cooling from the liquid crystalline phase results in (or is accompanied by) the change of the lipid polar head and interface groups, with partial break of the intermolecular hydrogen bonding NH3þ 3 3 3 OdC.62 We thus suggest that the lack of the intermolecular attractive forces (such as hydrogen bonding and Langmuir 2009, 25(23), 13394–13401
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attractive electrostatic interactions) between the neighboring polar headgroups of DODAB molecules may explain the occurrence of the nonsynchronicity phenomenon during the liquid crystalline to coagel phase transition process.
4. Conclusions We have studied in detail the thermotropic phase behavior of the DODAB aqueous dispersions by using DSC, time-resolved FTIR, and XRD techniques. The stable phases at high and low temperatures are liquid crystalline and coagel phases, respectively. Upon heating, the coagel phase converts to the liquid crystalline phase, while upon cooling, the liquid crystalline phase converts to the coagel phase via the metastable gel phase. The intermediate gel phase has been captured and better characterized by a fast cooling (5 °C/min) experiment. In the viewpoint of molecular mechanisms, transformation from liquid crystalline to coagel phase upon cooling involves both an orderly rearranging process in the hydrocarbon tails and a dehydration process in the headgroup region. Most interestingly, we found that the head and tail regions of DODAB do not change synchronously during the process. The appearance of the metastable or intermediate gel phase separates the overall transformation process into two distinct stages (liquid crystalline to gel and gel to coagel). The first stage is characterized with marked change in the conformational order and packing state of the lipid tails,
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without detectible changes in the conformational and hydration properties of the DODAB headgroups. The second stage, on the other hand, is characterized by both the dehydration process of the lipid headgroups and the further rearrangements of the lipid tails. That is, the lipid tails change prior to the headgroups during the liquid crystalline to coagel transition. This time lag of the change of headgroups during cooling is the reason for the occurrence of the intermediate gel phase. It is suggested that the lack of the intermolecular attractive forces between the neighboring polar headgroups of amphiphiles may be the origin of the nonsynchronicity phenomenon. Such nonsynchronicity phenomenon in the self-assembled aggregates composed of the mediumsized lipid molecules reflects the regional (head/tail) imbalance in molecular interactions. These findings will not only provide new insight into the formation mechanism of the coagel phase from the liquid crystalline phase but will also shed light on our understanding of the polymorphism and reversibility of lipids including biomembrane phospholipids. Acknowledgment. This work was supported by grants from the Natural Science Foundation of China (NSFC: 20633080) and a National Basic Research Program of China (Grant No. 2006CB806200). The SAXS data were collected at the beamline 1W2A of the Beijing Synchrotron Radiation Facility (BSRF) with the assistance of Dr. Zhong-Hua Wu and Zhi-Hong Li.
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