Phase Behav
ior, Functions, and Medical Applications of Soy Phosphatidylcholine
and D
iglyceride Lipid Compositions
Fredrik Tiberg,*1,2Markus Johnsson,1Marija Jankunec,3and Justas Barauskas3,4 1Camurus AB, Ideon Science Park, Gamma Building, Sölvegatan 41, SE-22370 Lund, Sweden
2Physical Chemistry, Lund University, P. O. Box 124, SE-22100 Lund, Sweden 3Vilnius University Institute of Biochemistry, Mokslininkų 12, LT-08662 Vilnius, Lithuania 4Biomedical Science, Faculty of Health and Society, Malmö University, SE-20506 Malmö, Sweden
(Received May 14, 2012; CL-120511; E-mail: Fredrik.Tiberg@camurus.com) Lipid compositions with the ability to self-assemble into
biocompatible nano- and mesostructured functional materials have many potential uses in modern medicine. By using two-component lipid systems, it is possible to tune the structure formation and related functional properties, e.g., the encapsula-tion and extended release of small molecules and peptides, by simply varying the ratio of the lipid building blocks. This is shown in detail for the binary phosphatidylcholine and diglyceride lipid systems, which are currently being used in multiple programs for the development of novel pharmaceuticals and marketed products.
The ability of certain lipids to self-assemble into functional reversed-phase nonlamellar liquid crystal (LC) gels in contact with aqueous media, make such systems highly interesting for use as ambient responsive delivery systems using such func-tional features as bioadhesion, biodegradation, encapsulation, and controlled release.1 By exploiting the liquid-to-LC gel transition triggered on exposure of lipid solutions to aqueous media, it is possible to combine excellent in vivo encapsulation and extended release properties of some reversed LC gel phases, with the easy manufacturing and administration properties of relatively low-viscosity nonaqueous lipid solutions comprising small amounts of nonaqueous solvent.1,2
The mostfrequently used nonlamellar LC-forming lipid in drug delivery-related studies has been glycerol monooleate (GMO).3,4 At physiological conditions in excess water, GMO forms a bilayer-based bicontinuous cubic phase (V2) with the Pn3m space group representing a three-dimensional network of hydrophilic and hydrophobic domains. Although a promising candidate for several applications, GMO-based LCs have been shown to exhibit a pronounced tendency to disrupt membrane structures, e.g., extensive hemolytic activity.5Furthermore, the one-component GMO system hasfurther limitations with respect to the drug delivery application in the difficulty of compensating for any phase changes caused by solubilizing drug compounds.6 The use of a two or multicomponent system can typically alleviate this issue and can allow for compensation of unwanted phase changes by simply adjusting the ratio of the lipid building blocks. One example of such a system is the two-component unsaturated (e.g., dioleoyl) phosphatidylcholine (PC) and glyc-erol dioleate (GDO) system, where PC has a preference for the planar lamellar LC phase (L¡) and GDOfor the reversed liquid micellar phase (L2). Between these extremes at full hydration reversed 2D hexagonal (H2), reversed micellar cubic (I2) with the Fd3m space group as well as two- and three-phase regions areformed.711
A recent study on the in vitro release of disodium fluorescein from soy PC (SPC)/GDO nonlamellar LCs has shown that the minimum of release (lowest release rates) can be found for compositions in the two-phase region between the H2 and I2 phases.1 Importantly, the observed release of both for small molecules1 and peptides2 from these phases are signifi-cantly more restricted compared with, e.g., corresponding release rates from the highly viscous GMO V2 phase; this shows theimportance of phase morphology and the usefulness of H2 and I2 phases of the SPC/GDO system in long-acting delivery applications. Indeed, the pharmacokinetic (PK) profile obtained after the injection (rat) of SPC/GDO-based formulation comprising somatostatin 114 shows a stable and dose-propor-tional plasma concentration of this peptide for over one week, despite a circulation half-life of only a few minutes.1,12Another example illustrating the unique long-acting in vivo release properties of SPC/GDO formulations was recently provided for the peptide leuprolide acetate.2 When benchmarked against leading commercial injection depot products based on poly-(lactic-co-glycolactic) (PLGA) polymers, the SPC/GDO system wasfound to provide both a lower initial release (low “burst” release) and more consistent plasma leuprolide levels over time.2 Here, we have studied in detail the equilibrium aqueous-phase behavior and aqueous-phase structures for the two-component SPC and GDO system as a function of lipid composition by using synchrotron small-angle X-ray scattering (SAXS) at the I711 and I911-4 beamlines at MAX-lab (Lund University, Sweden). Details of sample preparation and SAXS experiments are described in Supporting Information (SI).13
Figure 1a presents the measured SAXS profiles of fully hydrated mixtures of SPC/GDO between lipid ratios of 70:30 and 20:80 by weight at every 2.5 wt %. Figure 1b shows the SAXS data of fully hydrated SPC/GDO mixtures in a more narrow composition region between 42:58 and 35:65 at every 1 wt%.
As seenfrom SAXS data in Figures 1a and 1b, the phase behavior of SPC/GDO is indeed more rich and complex than the reported phase sequence for DOPC/GDO system in excess water conditions. At a low GDO content and up to SPC/GDO weight ratio of 62.5:37.5, a 2D H2 phase is formed that is characterized by five distinctive reflections at relative positions in ratios of 1:pffiffiffi3:2:pffiffiffi7:3.14The calculated lattice parameter (a) for 2D H2phase decreasesfrom 7.66 to 7.25 nm between SPC/ GDO ratio of 70:30 and 62.5:37.5. With increasing GDO content, a cubic phase starts to emerge, which is a one-phase region that exists between SPC/GDO ratios of 50:50 and 45:55. This is clearly shown by the location of the relative positions in ratios of the first 9 Bragg peaks, i.e., pffiffiffi3:pffiffiffi8:p11ffiffiffiffiffi:pffiffiffiffiffi12:pffiffiffiffiffi16: Published on the web October 20, 2012 1090
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ffiffiffiffiffi 19 p
:pffiffiffiffiffi24:pffiffiffiffiffi27:pffiffiffiffiffi32, which can be indexed as the reflections of aface-centered I2phase of the Fd3m space group (Figure 2a). With increasing GDO content, the obtained a value for this phase varies from 18.7 to about 17.3 nm, related to the increasing negative curvature and/or decreasing swelling ability of the cubic phase. At even higher GDO concentration, an extremely narrow region of another LC phase is located between SPC/GDO weight ratios of approximately 42:58 and almost 40:60 (Figure 2b). 20 Bragg peaks were identified, and their positions and indexing are summarized in SI.13 Indexing of SAXS data of this phase was achieved by assuming 3D hexagonal close packing (hcp) structure. The following relation was used where the dhklis related to the two lattice parameters, a and c, and where R= c/a:
1 dhkl2¼ 1 ahkl2 3 4ðh2þ k2þ hkÞ þ l2 R2 ð1Þ The analysis clearly shows that the structure of this phase is consistent with the hcp lattice of identical spherical reversed micelles having the P63/mmc symmetry. The calculated lattice parameters for this 3D hexagonal phase are a = 6.99 and c= 11.35 nm with a c/a ratio of 1.624, which is very close to the value for the ideal hcp lattice of identical spheres, Rffiffiffi ideal=
8 p
:3= 1.633. In addition, the intensities of obtained SAXS reflections are similar to those obtained for the 3D hexagonal P63/mmc phase of other lipid15 and surfactant16 systems in water.
Between SPC/GDO weight ratios of approximately 39:61 and 37:63, a very narrow region of yet another LC phase was identified. SAXS data of this phase is shown in Figure 2c. It shows atleast 30 Bragg reflections that are not consistent with cubic or 3D hexagonal lattices of neighboring phases, and we were unable to resolve its structure. We can only speculate that since it is between the P63/mmc and Fd3m phases, its structure should be “intermediate” between hexagonally close-packed identical micelles and the cubic Fd3m lattice consisting of two types of reversed micelles.
Starting from the SPC/GDO ratio of 35:65, the cubic Fd3m phase that shows a very nice SAXS pattern with at least 15 Bragg peaksis found again (Figure 2d). With increasing GDO content, the calculated a value for this phase decreases from about 15.81 to 15.42 nm before transforming into a reversed micellar phase (L2) at a SPC/GDO weight ratio of 20:80.
The intricate phase behavior of fully hydrated SPC/GDO mixtures described here has not been observed before. Most of the studies were performed with mixtures of dioleoylphospha-tidylcholine (DOPC)/GDO, which shows a less rich phase behavior.810 Our results (see SI) confirm that DOPC/GDO mixtures only form the H2 and Fd3m phases in excess-water conditions. The existence of the P63/mmc phase was not demonstrated for the pure DOPC/GDO (at a weight ratio of 40:60) system at limiting hydration.17 The formation of 3D hexagonal phase was only shown for the ternary DOPC/GDO/ cholesterol system.14Here,it should also be noted that contrary to our findings, the previously determined phase behavior of fully hydrated SPC/GDO also shows no formation of the P63/ mmc and“intermediate” unknown phases.11The reasonfor this difference is that the authors in that study used D2Oinstead of H2O. As shownin SI, the phase behavior of SPC/GDO, even in a mixture of H2O/D2O (80:20 wt/wt), is dramatically different
(a)
(b)
Figure 1. SAXS profiles of fully hydrated mixtures of SPC/ GDO as a function of lipid composition between SPC/GDO weight ratios of 70/30 and 20/80 (a), and 42/58 and 35/65 (b) at 25 °C.
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with the formation of only the H2and Fd3m phasesin the lipid mixture composition region.
In conclusion, fully hydrated SPC/GDO mixtures with increasing GDO content forms the following phase sequence: L¡¼ H2¼ Fd3m ¼ P63/mmc ¼ unknown “intermediate” ¼ Fd3m¼ L2. Note that the reappearance of the Fd3m phase at a different GDO content is not surprising and does not contradict the Gibbs phase rule. These two phases are indeed intercon-nected atlimited hydration values (see SI), and they form a one-phase region. Our findings show that the two-component diacyl lipid system is rich in phase behavior, and as shown in previous studies,1,2the system presents a high potential for encapsulation andlong-acting release of small molecules and peptides, which are emphasized by the current portfolio of drug development programsin early-to-late clinical stages.18
Paper based on a presentation made at the International Association of Colloid and Interface Scientists, Conference (IACIS2012), Sendai, Japan, May 1318, 2012.
References and Notes
1 F. Tiberg, M. Johnsson, J. Drug Delivery Sci. Technol. 2011, 21, 101.
2 F. Tiberg, M. Johnsson, C. Nistor, F. Joabsson, in Long Acting Injections and Implants in Advances in Delivery Science and Technology, ed. by J. C. Wright, D. J. Burgess, CRS-Springer, New
York, 2012, pp. 315333.doi:10.1007/978-1-4614-0554-2_16. 3 J. C. Shah, Y. Sadhale, D. M. Chilukuri,Adv. Drug Delivery Rev.
2001, 47, 229.
4 C. J. Drummond, C. Fong, Curr. Opin. Colloid Interface Sci. 1999, 4, 449.
5 J. Barauskas, C. Cervin, M. Jankunec, M. Špandyreva, K. Ribokaitė, F. Tiberg, M. Johnsson,Int. J. Pharm. 2010, 391, 284. 6 S. Engström, L. Engström,Int. J. Pharm. 1992, 79, 113. 7 S. Das, R. P. Rand,Biochem. Biophys. Res. Commun. 1984, 124,
491.
8 S. Das, R. P. Rand,Biochemistry 1986, 25, 2882. 9 J. M. Seddon,Biochemistry 1990, 29, 7997.
10 V. Luzzati, R. Vargas, A. Gulik, P. Mariani, J. M. Seddon, E. Rivas,Biochemistry 1992, 31, 279.
11 G. Orädd, G. Lindblom, K. Fontell, H. Ljusberg-Wahren,Biophys. J. 1995, 68, 1856.
12 C. Cervin, P. Vandoolaeghe, C. Nistor, F. Tiberg, M. Johnsson, Eur. J. Pharm. Sci. 2009, 36, 377.
13 Supporting Information is available electronically on the CSJ-Journal Web site, http://www.csj.jp/journals/chem-lett/index.html. 14 At SPC/GDO weight ratios of 70/30 and 67.5/22.5 small peaks observed atlow q may be indexed as traces of bicontinuous cubic Pn3m and Im3m phases with a = 16.5 and 21 nm¹1, respectively.
15 G. C. Shearman, A. I. I. Tyler, N. J. Brooks, R. H. Templer, O. Ces, R. V. Law, J. M. Seddon,J. Am. Chem. Soc. 2009, 131, 1678. 16 X. Zeng, Y. Liu, M. Impéror-Clerc,J. Phys. Chem. B 2007, 111,
5174.
17 J. M. Seddon, J. Robins, T. Gulik-Krzywicki, H. Delacroix,Phys. Chem. Chem. Phys. 2000, 2, 4485.
18 Camurus AB, Lund, Sweden, 2012. http://www.camurus.com Figure 2. SAXS profiles of the LC phases of fully hydrated mixtures of SPC/GDO as a function of lipid composition at 25 °C SPC/ GDO weight ratios are: 47.5/52.5 (a), 40/60 (b), 37.5/62.5 (c), and 35/65 (d).
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