Langmuir 2000, 16, 7355-7358
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13
C NMR Study of Aqueous Dispersions of Reversed Lipid Phases
Maura Monduzzi,* Helena Ljusberg-Wahren,† and Kåre Larsson† Dipartimento di Scienze Chimiche, Cittadella Universitaria Monserrato, S.S. 554 Bivio per Sestu, 09042 Monserrato-CAGLIARI, Italy, and Camurus Lipid Research, Ideon, Gamma 1, Solvegatan 41, SE-223 70 LUND, Sweden Received January 24, 2000. In Final Form: April 25, 2000 Monoolein (GMO) forms a bicontinuous cubic phase that can coexist in equilibrium with a water phase, and this phase can be dispersed into colloidal particles. Poloxamer is an efficient stabilizer for dispersions of this phase. By adding a small amount of triolein to GMO, a reversed hexagonal phase is formed at room temperature. This phase can be dispersed into submicron particles in water in the same way. The inner molecular arrangement of these dispersions was investigated by 13C NMR relaxation. The 13C NMR relaxation rates of the various GMO carbon atoms obtained from the dispersed particles compared to those obtained from the nondispersed phases indicate that the lipid organization and dynamic properties of the original nondispersed phases are retained. It is also demonstrated that the cubic phase is transformed into the reversed hexagonal phase, within six months, due to hydrolysis of GMO. A remarkable observation is that no significant hydrolysis occurs during this time in the corresponding dispersed samples. This indicates that solubilization of a few wt % of Poloxamer into the GMO bilayer provides protection against degradation.
1. Introduction Colloidal particles obtained by dispersion of cubic and reverse hexagonal liquid crystalline (L. C.) phases have recently been obtained and characterized by cryo-TEM and X-ray diffraction.1,2 The cubic and hexagonal liquid crystals were prepared from the nonionic lipid glycerolmonooleate (GMO), and both are reversed phases according to the curvature of the bilayer. At room temperature, GMO forms, with water, only lamellar and cubic liquid crystals.3,4 The hexagonal liquid crystals can be obtained easily by adding 6-12 wt % of triolein (GTO) to GMO. Aqueous stable dispersions of these reversed phases have been obtained using a nonionic tribloc polymer (Polaxamer 407) as stabilizing agent.1,2 Landh5 reported phase equilibria in the ternary phase diagram glycerol monooleate (GMO)-water-Poloxamer 407 and also described the possibility of producing dispersions of cubic phases that occur in this system. Polaxamer 407 (P)swith molecular formula PEO98PPO67PEO98sis more hydrophobic in the PPO region compared to the hydrophilic PEO regions and can therefore be solubilized into lipid bilayers. The more hydrophilic PEO ends protrude into the aqueous medium, thus giving a steric stabilization. Electron microscopy has shown that the shape of the submicron particles of one kind of cubic phase is prefer* To whom correspondence should be addressed at Cittadella Universitaria Monserrato. Tel.: (39) 070675 4385. Fax: (39) 070675 4388. E-mail:
[email protected]. † Camurus Lipid Research. (1) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611. (2) Gustafsson, J.; Ljusber-Wahren, H.; Almgren, M.; Larsson, K., Langmuir 1997, 13, 6964. (3) Larsson, K. Nature 304, 1983, 664. (4) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213. (5) Landh, T. J. Phys. Chem. 1994, 98, 8453.
entially cubic with edges parallel to the axes of the unit cell. SAXS measurements of these dispersed particles have suggested the occurrence of a body-centered CP type of cubic arrangement (space group Im3m). This cubic structure is not found in GMO/W cubic phases and is thus a consequence of solubilized Poloxamer. Indeed, in the GMO/W system the cubic gyroid CG (space group Ia3d) and the cubic diamond CD (space group Pn3m) liquid crystalline (L. C.) phases have been identified earlier.3 The particle shapes have been confirmed by atomic force microscopy (AFM) in a very recent study.6 These dispersed systems have found some promising applications in the field of fully biocompatible drug delivery systems7 and also as enzyme carriers for the food industry.1,2 Thus, it is important to investigate the longterm stability of the lipid matrixes. The aim of the 13C NMR relaxation measurements reported here was to examine the inner microstructure of the dispersed particles compared to the corresponding nondispersed phases. 13C NMR is a powerful technique with which to follow the molecular arrangement in the bilayer and its dynamic. A few papers have reported NMR data on GMO systems.8-11 2. Experimental Section The preparation of GMO we used is a distilled monoglyceride (RYLO MG 90, Danisco Ingredients, Brabrand, Denmark) with the following fatty acid composition: oleic acid, 92 wt %; linoleic (6) Neto, C.; Aloisi, G.; Baglioni, P.; Larsson, K. J. Phys. Chem. B 1999, 103, 3896. (7) Preliminary results on GMO/Adamantane/Water systems have shown different release rates of the additive from cubic and reverse hexagonal L.C phases. (8) Eriksson, P.-O.; Lindblom, G. Biophys. J. 1993, 129. (9) Caboi, F.; Nylander, T.; Razumas, V.; Talaikyte´, Z.; Monduzzi, M.; Larsson, K. Langmuir 1997, 13, 5476. (10) Pampel, A.; Strandberg, E.; Lindblom, G.; Volke, F. Chem. Phys. Lett. 1998, 287, 468. (11) Caboi, F.; Amico, G. S.; Pitzalis, P.; Monduzzi, M.; Nylander, T.; Larsson, K. Chem. Phys. Lipids, 2000, submitted for publication.
10.1021/la0000872 CCC: $19.00 © 2000 American Chemical Society Published on Web 08/16/2000
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acid, 6 wt %; and saturated acids, 2 wt %. The glycerol trioleate (GTO) used is a high monosaturated sunflower oil (Trisun 80 SVO, Eastlake, Ohio). Polaxamer 407 (P) was obtained from BASF (Germany). Samples of the GMO/W binary systems were prepared by weighing appropriate amounts of the lipids into glass tubes (φ ≈ 0.5 cm), melting them at 40 °C, and then adding water. Samples were centrifuged at 2800-3000 rps and 39 °C, frozen for 12 h, then flame-sealed and homogenized by repeated cycles of heating and centrifuging. They were then stored at 25 °C for at least 3 weeks or until the samples were homogeneous. A hexagonal phase was formed in a sample prepared in the cubic region of the GMO/W phase diagram when it had been stored at room temperature for 6 months. A homogeneous birefringence characteristic for reversed hexagonal phases was observed in the polarizing microscope, and small-angle X-ray scattering (SAXS) measurements confirmed the occurrence of a hexagonal phase. The dispersion procedure has been reported earlier.1,2 The colloidal particle dispersion composition is 5 wt % of a cubic or hexagonal phase and 95 wt % of water. The lipid phase consists of 94 wt % of the GMO/W or GMO/GTO/W L. C. phase and 6 wt % of P. The hexagonal phase was prepared at two different GMO/ GTO ratios: 96/4 and 88/12. All samples were stored in the dark at 25 °C. 13C NMR measurements were performed by a Varian VXR400 (9.4 T) spectrometer at the operating frequency of 100.57 MHz at 25 °C. A standard variable-temperature control unit, with an accuracy of (0.5 °C, was used. The 13C NMR spin-lattice relaxation times (T1) were measured by the standard inversion recovery sequence (PD-180-τ-90-AC) by acquiring the partially relaxed spectra at 12-14 different τ values. The T1 relaxation times were obtained by three-parameter nonlinear fitting:
I(τ) ) I(0) exp(-τ/T1) The error on the fitting was always less than 1%, and the reproducibility of the T1 values was within (5%.
3. Results and Discussion The characteristic shapes of the dispersed particles of the cubic and hexagonal L. C. phases, having a similar composition to that in this work, were obtained by cryo transmission electron microscopy1,2 and are shown in Figure 1. The lipid bilayer is resolved, and it can be seen that the whole particle is a single crystal. An earlier SAXS examination2 of the cubic particles exhibited strong line broadening, but it could still be concluded that the symmetry corresponds to the CP phase (space group Im3m) and the dimensions were in agreement with those of the corresponding nondispersed phase.5 The line broadening can be due to the small particle size and/or disorder within the lipid bilayer structure. As will be shown below, however, the 13C NMR data indicate that the organization and the dynamics of the bilayer are retained during the dispersion process. Figure 2 shows the 13C NMR spectra of the cubic CG (Figure 2a) and the reversed hexagonal HII (Figure 2b) phase of the same GMO/W sample containing 27 wt % water. The spectra were recorded after 1 month and after 6 months from the time of sample preparation. The identification of the phases was based on birefringence and was confirmed by SAXS. NMR spectra of isotropic L. C. phases can generally be obtained with high resolution, provided that the sample is uniform (without bubbles) inside the NMR tube. In the case of an anisotropic L. C. phase, such as a lamellar or a hexagonal phase, two preferential orientations (0° and 90°) of the symmetry axis with respect to the magnetic field direction can be achieved by allowing the sample to cool slowly inside the magnet. This procedure is generally used whenever a quadrupolar nucleus (spin quantum
Figure 1. Dispersion of a GMO/P/W cubic phase (above) and a GMO/GTO/P/W reverse hexagonal phase (below) as seen in cryo-TEM,1,2 reproduced by permission. The cubic phase is a CP phase with edges of the particles along the cubic axes, and the inner periodicity has a repetition distance of about 10 nm. The largest particle of the hexagonal phase is seen along the c-axis, and the bar in this micrograph is 100 nm.
number I g 1) is being observed. The alignment of the L. C. structure improves the observation of the NMR quadrupolar splitting. This is not the case with 13C (I ) 1/2). The procedure was, however, used as it provides a better resolution of the NMR signals, particularly in the HII phase. Comparison of the two spectra demonstrates the occurrence of GMO hydrolysis that results in the CG f HII phase transition. Indeed, the NMR signals due to free glycerol can be clearly identified in Figure 2b. The presence of free oleic acid is indicated by the appearance of two broad signals at low field due to the different chemical shifts of the GMO and the carboxylic acid -CO- groups. The -CHdCH- double bond group gives an NMR signal that is shaped according to a chemical shift anisotropy (CSA) effect. To our knowledge, CSA for spin I ) 1/2 has been reported only for the 31P NMR signals of phospholipids and for the 19F and 133Cs NMR signals of a surfactant counterion in lamellar and hexagonal L. C. phases.12 Here, CSA is likely to be due to the two different alignments of the double-bond directory with respect to the magnetic (12) Lindblom, G. In Advances in Lipid Methodology, Part III; Christie, W. W., Ed.; The Oily Press Ltd.: Dundee, 1996; Vol. 7, Chapter 5; p 133.
Aqueous Dispersion of Reversed Lipid Phases
Figure 2. 13C NMR spectra of a GMO/W (composition 73/27 wt/wt) sample recorded at different times from the sample’s preparation: (a) at 1 month, when sample is in the cubic CG phase. The attribution of the NMR signals of relevant GMO carbons is also reported; (b) at 6 months, when sample has evolved to a reverse hexagonal HII phase. G1*, G2*, and G3* indicate the 13C NMR signals of free glycerol.
field in the anisotropic L. C. phase. A similar effect may also be expected for the -CO- group. However, due to broadening and low intensity, it cannot be determined whether the two small peaks due to GMO and free oleic acid show any CSA phenomena. Another interesting feature of the spectrum given in Figure 2b is the large broadening of the G3 carbon peak from glycerol. The latter carbon and the -CO- carbons are expected to be strongly influenced by the change of curvature at the bilayer/water interface due to the CG f HII phase transition. This results in a compression of the polar group, which also implies a decrease of the mobility of the involved carbons. This, in turns, decreases the spin-spin relaxation time, T2, which is related to the line width by the approximated relation T2 ) 1/π∆ν1/2, where ∆ν1/2 is the width at halfheight of the NMR signal. Figure 3 shows the 13C NMR spectra of the dispersed systems: Figure 3a is the dispersion of the cubic phase and Figure 3b,c are the dispersion of the reversed hexagonal phase with a GMO/GTO ratio of 94/6 and 88/ 12, respectively. A comparison with the spectra reported in Figure 2 demonstrates that the main features of the molecular arrangement and the dynamics of the bilayer are retained when the L. C. phases are dipersed, as the chemical shifts are almost equal. The relatively higher intensities of the G2 and G1 signals in the spectra of the dispersions are due to the overlap with the -CH- and -CH2- signals of the dispersing polymer P. A new small signal, due to the different -CH3 groups of the PPO segment of the polymer P, appears at low field with respect to the lipid -CH3 groups. The presence of GTO can be deduced by the appearance of a new, rather broad C1 signal, due to the -CO-G2 group, which is located in the region 174.5-175 ppm. The -CO-G1/G3 signals retain the same chemical shift, δ ) 176.5 ppm, as in GMO. The oleic chain carbons exhibit the same chemical shifts, independent of the ester bond position in the glycerol group. The spectra of the dispersed hexagonal phases do not
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Figure 3. 13C NMR spectra of liquid crystal dispersions (5 wt % of dispersed phase in water). The ratio (lipid L. C. phase)/ (stabilizing agent P) is 94/6 wt/wt). Spectra were recorded 8 months after sample preparation: (a) GMO based cubic CP L. C. dispersion. “P” indicates the -CH- resolved signal due to the PPO region of Polaxamer 407; (b) GMO/GTO ) 96/4 based reverse hexagonal L. C. dispersion; and (c) GMO/GTO ) 88/12 based reverse hexagonal L. C. dispersion. In (b) and (c) spectra, P and G2 signals overlap; thus, the total intensity increases.
show any CSA effect for the chain double bond. This is not surprising. Indeed, in this case no preferential orientation in the magnetic field can be expected for such small colloidal particles. Only a general broadening of the NMR signals is observed. Again, a specific broadening of the G3 and -CO- group is observed in the dispersed hexagonal phase of Figures 3b and 3c, exactly as in the case of the nondispersed HII L. C. phase in Figure 2b. This indicates also that, within the limit of NMR detection, no free GMO molecules are released during the dispersion process. It is worth noticing that GMO hydrolysis products can hardly be observed in the dispersion of the cubic and hexagonal phases. Indeed, the dispersed samples used to record the 13C NMR spectra of Figure 3 were stored for 8 months under the same conditions as the HII sample of Figure 2b. The 13C NMR signal of the C1 carbon due to free oleic acid, expected around 180 ppm, does not appear either in the cubic or in the hexagonal dispersions. In addition, the similar intensity ratios between G2 and G1 signals of GMO, observed in Figure 3, together with a very small signal around 72 ppm due to the G2 carbon of free glycerol, confirm that hydrolysis of the monoglyceride is almost undetectable within the limits of the NMR technique. From a comparison of crystal lattices of dispersed and nondispersed phases,2 it was earlier concluded that the lipid bilayer in the core of the cubic dispersed particles contain above 2 wt % and below 6 wt % of P molecules. Evidently, this amount of solubilized P molecules protects the GMO molecules in the bilayer against degradation. An additional demonstration that the molecular arrangement and time-dependent interactions are equal in the liquid crystalline phases and in their dispersions is provided by 13C NMR relaxation data. Relaxation times are rather sensitive to modifications of the local intermolecular interactions and to molecular dynamics. Figure 4 shows the 13C NMR spin-lattice relaxation times, T1, measured for the CG cubic phases of GMO in water together with those measured for the cubic and hexagonal dispersions. The T1 values for each carbon atom of the glycerol polar head and the hydrocarbon chain are equal, within the experimental error, for all these structures. T1 for the
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the same mobility with the exception of the C16-C18 carbons. The latter carbons display mobilities that are typical of liquids. The presence of the double bond (C9C10) produces an immobilization of the neighboring carbons, as observed for those close to the polar-apolar interface. The intermolecular forces responsible for such dynamic effects result in the stabilization of the bilayer arrangement of the cubic phase and persist after the CG f HII phase transition. Most remarkable is the persistence, during the dispersion process, of the local interactions in the polar head region as well as along the hydrophobic tail of the GMO molecule. 4. Concluding Remarks 13
Figure 4. 13C NMR spin-lattice relaxation time T1 for the polar head and for the hydrophobic tail of GMO measured in various environments: (b) CG L. C. (GMO/W ) 68/32); (×) HII L. C. (GMO/W ) 73/27, 6 months old, cf. Figure 3b); (4) Cubic CP L. C. dispersion; (0) HII (GMO/GTO ) 94/6) L. C. dispersion; and (]) HII (GMO/GTO ) 88/12) L. C. dispersion. Dispersed samples composition as in Figure 3.
-CO- group could not be measured in the hexagonal environments, and it is not reported in Figure 4. As a general comment, it should be noticed that in moving from the polar head toward the end of the hydrophobic tail, the various carbons show approximately
C NMR studies of GMO/P/W cubic and GMO/GTO/ P/W reverse hexagonal phases demonstrate that molecular interactions and dynamic properties in these dispersed states are closely related to the nondispersed GMO/W L. C. phases. It was found that a minor concentration of polymer molecules solubilized in the hydrophobic domain of the L. C. phases provides efficient protection against hydrolysis. Acknowledgment. MURST(Italy), CNR (Italy), Consorzio Sistemi Grande Interfase (CSGI-Firenze), and Assessorato Igiene Sanita´ (Sardinia Region-Cagliari) are acknowledged for support. LA0000872
