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Solubilization of Hydrophobic Guest Molecules in the Monoolein Discontinuous QL Cubic Mesophase and Its Soft Nanoparticles Rivka Efrat,† Ellina Kesselman,‡ Abraham Aserin,† Nissim Garti,† and Dganit Danino*,‡ Casali Institute of Applied Chemistry, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel, and Department of Biotechnology and Food Engineering and the Russell Berrie Nanotechnology Institute, Technion;Israel Institute of Technology, Haifa 32000, Israel ReceiVed May 26, 2008. ReVised Manuscript ReceiVed July 22, 2008 Hydrophobic bioactive guest molecules were solubilized in the discontinuous cubic mesophase (QL) of monoolein. Their effects on the mesophase structure and thermal behavior, and on the formation of soft nanoparticles upon dispersion of the bulk mesophase were studied. Four additives were analyzed. They were classified into two types based on their presumed location within the lipid bilayer and their influence on the phase behavior and structure. Differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), polarized light microscopy, cryogenictransmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS) were used for the analysis. We found that carbamazepine and cholesterol (type I molecules) likely localize in the hydrophobic domains, but close to the hydrophobic-hydrophilic region. They induce strong perturbation to the mesophase packing by influencing both the order of the lipid acyl chains and interactions between lipid headgroups. This results in significant reduction of the phase transition enthalpy, and phase separation into lamellar and cubic mesophases above the maximum loading capacity. The inclusion of type I molecules in the mesophase also prevents the formation of soft nanoparticles with long-range internal order upon dispersion. In their presence, only vesicles or sponge-like nanoparticles form. Phytosterols and coenzyme Q10 (type II molecules) present only moderate effects. These molecules reside in the hydrophobic domains, where they cannot alter the lipid curvature or transform the QL mesophase into another phase. Therefore, above maximum loading, excess solubilizate precipitates in crystal forms. Moreover, when type II-loaded QL is dispersed, nanoparticles with long-range order and cubic symmetry (i.e., cubosomes) do form. A model for the growth of the ordered nanoparticles was developed from a series of intermediate structures identified by cryo-TEM. It proposes the development of the internal structure by fusion events between bilayer segments.
Introduction Lipid mesophases are potential sustained release vehicles for pharmaceuticals, cosmetic formulations, and food products because of their extensive capabilities of encapsulation of hydrophilic, hydrophobic, and amphiphilic bioactives, and the protection they provide to the encapsulated molecules.1-4 However, the guest molecules may significantly influence the bulk liquid crystalline phase behavior by inducing structural transformations into other geometries and topologies or by separation into two phases.5 The structural transitions depend on interactions between the lipid and the guest molecule, and the quantity entrapped.5-9 Certain compounds reduce the bilayer curvature. This was demonstrated recently with sodium diclofenac (Na-DFC), where increasing quantities of the solubilized drug in the discontinuous cubic QL mesophase induced transitions to * Corresponding author. E-mail:
[email protected]. Phone: +9724-829-2143. Fax: +972-4-829-3399. † The Hebrew University of Jerusalem. ‡ Technion;Israel Institute of Technology. (1) Fa, N.; Babak, V. G.; Stebe, M. J. Colloids Surf., A: Physicochem. Eng. Aspects 2004, 243(1-3), 117–125. (2) Yang, D.; Armitage, B.; Marder, S. R. Angew. Chem., Int. Ed. 2004, 43(34), 4402–4409. (3) Barauskas, J.; Johnsson, M.; Johnson, F.; Tiberg, F. Langmuir 2005, 21(6), 2569–2577. (4) Barauskas, J.; Johnsson, M.; Tiberg, F. Nano Lett. 2005, 5(8), 1615–1619. (5) Barauskas, J.; Misiunas, A.; Gunnarsson, T.; Tiberg, F.; Johnsson, M. Langmuir 2006, 22(14), 6328–6334. (6) Chang, C. M.; Bodmeier, R. Int. J. Pharm. 1997, 147(2), 135–142. (7) Shah, J. C.; Sadhale, Y.; Chilukuri, D. M. AdV. Drug DeliVery ReV. 2001, 47(2-3), 229–250. (8) Yaghmur, A.; de Campo, L.; Sagalowicz, L.; Leser, M. E.; Glatter, O. Langmuir 2006, 22(24), 9919–9927. (9) Li, S. J.; Yamashita, Y.; Yamazaki, M. Biophys. J. 2001, 81(2), 983–993.
a bicontinuous cubic phase and eventually to a lamellar phase.10,11 The rate of release of the encapsulated bioactives depends on the mesophase and bioactive structures, and on physicochemical interactions between these components.7,12-15 It can be modulated if sufficient knowledge is gained on the structure of the mesophase.7,12,15 The high viscosity of hexagonal and cubic (glass-like) mesophases often limits their use to specific applications such as periodontal, mucosal, vaginal, and short-acting oral and parenteral drug delivery.7 An attractive path to overcome this limitation is to disperse the bulk mesophases into small soft particles that keep the mesophase long-range order and retain some of the bulk material properties.4,16-19 However, dispersing cubic and hexagonal lipid systems into nanoparticles often requires strong and prolonged shear, and results in the formation of particles in a wide range of sizes (1-100 µm). Recently it became possible to prepare low-viscosity mesophases, and consequently to form dispersed particles, at low shear rates and short application (10) Efrat, R.; Aserin, A.; Garti, N. J. Colloid Interface Sci. 2008, 321(1), 166–176. (11) Efrat, R.; Shalev, D. E.; Hoffman, R. E.; Aserin, A.; Garti, N. Langmuir 2008, 24, 7590–7595. (12) Clogston, J.; Craciun, G.; Hart, D. J.; Caffrey, M. J. Controlled Release 2005, 102(2), 441–461. (13) Kumar, K. M.; Shah, M. H.; Ketkar, A.; Mahadik, K. R.; Paradkar, A. Int. J. Pharm. 2004, 272(1-2), 151–160. (14) Welin-Berger, K.; Neelissen, J. A. M.; Engblom, J. J. Controlled Release 2002, 81(1-2), 33–43. (15) Clogston, J.; Caffrey, M. J. Controlled Release 2005, 107(1), 97–111. (16) Boyd, B. J. Int. J. Pharm. 2003, 260(2), 239–247. (17) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13(26), 6964–6971. (18) Rosa, M.; Infante, M. R.; Miguel, M. D.; Lindman, B. Langmuir 2006, 22(13), 5588–5596. (19) Spicer, P. T.; Hayden, K. L.; Lynch, M. L.; Ofori-Boateng, A.; Burns, J. L. Langmuir 2001, 17(19), 5748–5756.
10.1021/la8016084 CCC: $40.75 2009 American Chemical Society Published on Web 09/10/2008
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Figure 1. Chemical structures of CBZ (A), CoQ10 (B), and various sterols (C). Sterol key: brassicasterol, RdCH3 and an additional double bond at C-22; campesterol, RdCH3; Chl, RdH; β-sitosterol, RdCH2CH3; stigmasterol, RdCH2CH3 and an additional double bond at C-22.
time, for example, by incorporating a hydrotrope such as an alcohol, and selecting appropriate dilution pathways.19 These methods also produce much smaller and more homogeneous particles in the range of 100-500 nm, with improved kinetic stability.3 Further progress was gained by using mixed amino acid surfactants, for example, arginine-N-lauroyl amide dihydrochloride (ALA) with sodium hydrogenated tallow glutamate (HS) in water.18 A different approach to the formation of nonlamellar nanoparticles is the use of dry powdered precursors produced by spray drying in the presence of starch and dextran.20 Dispersing and stabilizing agents are key components in such preparations.2 The most frequently used stabilizing agent is Pluronic F-127 (Poloxamer 407), a nonionic triblock copolymer composed of polyethylene oxide (PEO) and polypropylene oxide (PPO) (PEO98PPO67PEO98). Nakano et al.21 demonstrated that, at low Poloxamer 407 concentrations, most of the polymer adheres to the particle surface, providing steric shielding and stabilizing the colloid particles against aggregation and coalescence. Caseins or polysorbate 80 (P80) are also used as stabilizing agents.2,3 In addition to lower viscosity, reversed nonlamellar nanoparticles offer high interfacial area and therefore, theoretically, increased loading capacities.8,22 However, as in the bulk mesophase, solubilized bioactives can influence the internal order of the nanoparticles, their size distribution, stability, and release pattern. Recently we reported on the formation of a new inverted discontinuous mesophase in the monoolein-ethanol-water system. This mesophase, which was designated as QL, has a primitive cubic symmetry.23 It is transparent and has a low viscosity, making it an ideal system for solubilization of drugs and nutraceuticals,10 and for the formation of loaded soft nanoparticles. In the present study we explore the loading of lipophilic bioactive compounds in the QL mesophase and its dispersed nanoparticles. The bioactive compounds studied are carbamazepine (CBZ), coenzyme Q10 (CoQ10), and two sterols: cholesterol (Chl) and phytosterols (PSs, plant sterols). CBZ (5Hdibenz[b,f]azepine-5-carboxamide, Figure 1A) is an important antiepileptic agent and drug to treat trigeminal neuralgia belonging to the family of iminostilbenes.24-26 CoQ10 is a ubiquinone composed of 50-carbon polyisoprene with a terminal quinone domain (Figure 1B). It is an integral and essential part of (20) Spicer, P. T.; Small, W. B.; Lynch, M. L.; Burns, J. L. J. Nanopart. Res. 2002, 4(4), 297–311. (21) Nakano, M.; Sugita, A.; Matsuoka, H.; Handa, T. Langmuir 2001, 17(13), 3917–3922. (22) Esposito, E.; Cortesi, R.; Drechsler, M.; Paccamiccio, L.; Mariani, P.; Contado, C.; Stellin, E.; Menegatti, E.; Bonina, F.; Puglia, C. Pharm. Res. 2005, 22(12), 2163–2173. (23) Efrat, R.; Aserin, A.; Kesselman, E.; Danino, D.; Wachtel, E. J.; Garti, N. Colloids Surf., A: Physicochem. Eng. Aspects 2007, 299(1-3), 133–145. (24) Vijay, T.; Anilkumar, H. G.; Yathirajan, H. S.; Narasimhamurthy, T.; Rathore, R. S. Acta Crystallogr., Sect. E: Struct. Rep. Online 2005, 61, O3718– O3720. (25) Kogan, A.; Aserin, A.; Garti, N. J. Colloid Interface Sci. 2007, 315(2), 637–647. (26) Kogan, A.; Popov, I.; Uvarov, V.; Cohen, S.; Aserin, A.; Garti, N. Langmuir 2008, 24(3), 722–733.
Table 1. Maximum Solubilization Capacity of the Guest Molecules in the QL Mesophase loaded guest molecule
maximum loading (mol %)
maximum loading (wt %)
CoQ10 CBZ PS Chl
0.02 0.05 0.20 0.40
0.50 0.35 2.45 4.78
mitochondrial ATP synthesis, carrying electrons and protons in oxidative phosphorylation and in other proton-pumping processes in the membranes. Since all of the organelle membranes are exposed to H+ gradients, they may also serve as inhibitors of proton leakage.27-29 CoQ10 (a yellow, water-insoluble crystalline powder) is an antioxidant used daily as a nutritional supplement and in the treatment of cardiovascular disorders such as angina pectoris, hypertension, and congestive heart failure.30 PSs belong to the triterpene family of natural products, which includes over 200 different sterols. They are a mixture of sterols structurally similar to Chl but with the inclusion of an additional side hydrophobic carbon chain at the C-24 position (Figure 1C). Sterols can be esterified to form the corresponding fatty acid esters (PS esters), and both classes are recognized as Chl-reducing agents. PSs act in the digestive track and are not transported to the blood stream; therefore they exhibit virtually no side effects and no mutagenic activity or subchronic toxicity in animals. Free natural PSs are hydrophobic, poorly soluble in food-grade oils and in water compounds, which limits their use in water-based applications such as clear beverages.31 The location of the solubilized molecules within mesophases (lamellar and QL) having the same composition was studied as a function of temperature. We also examined the loading capacity and the effect of the guest molecules on the phase behavior. The QL mesophase was dispersed into nanoparticles, and the effect of bioactive type on the structure and size of the formed nanoparticles was analyzed. These parameters were characterized using differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), polarized light microscopy, and cryogenic-transmission electron microscopy (cryo-TEM).
Experimental Section Materials. Distilled monoolein (distilled glycerol monooleate, GMO) consisting of 97.1 wt % monoglyceride, 2.5 wt % diglyceride (acid value 1.2, iodine value 68.0, melting point 37.5 °C), and 0.4 (27) Hauss, T.; Dante, S.; Haines, T. H.; Dencher, N. A. Biochim. Biophys. Acta: Bioenerg. 2005, 1710(1), 57–62. (28) Bhandari, K. H.; Newa, M.; Kim, J. A.; Yoo, B. K.; Woo, J. S.; Lyoo, W. S.; Lim, H. T.; Choi, H. G.; Yong, C. S. Biol. Pharm. Bull. 2007, 30(6), 1171–1176. (29) Aranda, F. J.; Go´mez-Ferna´ndez, J. C. Biochim. Biophys. Acta 1985, 820(1), 19–26. (30) Lenaz, G.; Fato, R.; Di Bernardo, S.; Jarreta, D.; Costa, A.; Genova, M. L.; Castelli, G. P. Biofactors 1999, 9(2-4), 87–93. (31) Rozner, S.; Aserin, A.; Wachtel, E. J.; Garti, N. J. Colloid Interface Sci. 2007, 314(2), 718–726.
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Figure 2. (A) Heating thermograms of QL and of QL loaded with equimolar quantities of CBZ, Chl, PS, and CoQ10. For clarity, the plots are shifted up in the y-axis. (B) X-ray diffraction patterns of empty QL and QL-loaded samples containing equimolar quantities of CBZ, Chl, PS, or CoQ10.
wt % free glycerol was obtained from Riken Vitamin Co., Ltd., Japan. Ethanol was analytical reagent grade (>99%), purchased from Frutarom, Ltd. (Haifa, Israel). Commercial CBZ (Figure 1A) was obtained from Teva Pharmaceutical Industries, Ltd. (Kfar Saba, Israel). PS (composed of 40-58 wt% β-sitosterol, 20-30 wt % campesterol, 14-22 wt % stigmasterol, and 0-6 wt % brassicasterol) was obtained from ADM Nutraceuticals (Decatur, IL). Chl (98% purity) was purchased from Sigma Chemical Co. (St. Louis, MO). CoQ10 was purchased from Kaneka Corporation (Osaka, Japan). Pluronic F-127 (PEO99PPO67PEO99) was a gift from BASF (Ludwigshafen, Germany). All ingredients were used without further purification. Water was double distilled. QL Mesophase Preparation. QL samples were prepared by weighing appropriate amounts of GMO, ethanol, and water, at a wt % ratio of 41:11:48 into culture tubes sealed with Viton-lined screw caps. The samples were heated to 60 °C for 2 min, stirred, and cooled to 25 ( 0.5 °C. The samples were allowed to equilibrate for 24 h before examination. Soft Nanoparticle Preparation. The method used to prepare soft nanoparticles has two steps. First, the QL mesophase (empty or loaded with the examined bioactive) was formed. Next, emulsification was performed at room temperature by adding this solution to an F-127 aqueous phase, yielding a final 10 wt % lipid solution. Soft nanoparticles were created upon mixing and shearing with the Ultra Turrax homogenizer (model T25, Janke & Kunkel, IKA Labortechnik, Staufen, Germany) at 3000 rpm for 20 min at room temperature. Methods. Differential Scanning Calorimetry. DSC measurements were used to characterize the thermal behavior and interaction of the guest molecules with the lipid structure. Experiments were carried out on a Mettler Toledo DSC822 instrument. Materials (5-15 mg) were weighed in standard 40 mL aluminum pans using a Mettler M3 microbalance and immediately sealed by a press. The samples were rapidly cooled at predetermined quenching rates, from ambient temperature to -10 °C. Each sample remained at this temperature for 20 min, and was then heated to 40 °C at a rate of 2 °C/min. The cooling/heating cycle was repeated twice. An empty pan was used as a reference. The fusion temperatures of the solid components and the total heat transferred in any of the observed thermal processes were determined. The enthalpy changes, associated with thermal transitions, were obtained by integrating the area of each pertinent DSC peak. The DSC temperatures reported here were reproducible to ( 0.5 °C. Small-Angle X-ray Scattering. SAXS measurements were used to identify the structure and the degree of internal order of the bulk mesophases and dispersed nanoparticles. Experiments were performed using Ni-filtered Cu KR radiation (1.540 Å) from an Elliott GX6 rotating anode operating at 1.2 kW. X-ray radiation was further monochromated and collimated by a single Franks mirror and then collimated by a series of slits and height limiters. The scattering profile was measured by a linear position-sensitive detector of the delay line type and stored on a PC as a 256-channel histogram. The sample-to-detector distance was approximately 48 cm. Each sample was sealed in a quartz capillary tube (diameter 1.5 mm) placed in a thermostatic bath. Measurements were carried out at 25 ( 0.5 °C
with exposure time of 1 to 3 h for the bulk mesophase, and 3 to 9 h for the nanoparticles. Cryogenic-Transmission Electron Microscopy. Cryo-TEM was used to identify the type of nanoparticles that form in the dispersed systems and study their internal structure. Specimens were prepared in a controlled environment vitrification system (CEVS)32 at 25 °C and 100% relative humidity. A 7 µL drop of each solution was placed on a TEM copper grid covered with a perforated carbon film (Pelco International, Clovis, CA) and blotted with filter paper to form a thin liquid film of the sample. The thinned sample was allowed to relax on the grid for 10 to 30 s, then plunged into liquid ethane at its freezing temperature to form a vitrified specimen, and transferred to liquid nitrogen for storage. The vitrified specimens were examined in a Tecnai T12 G2 transmission electron microscope (FEI) operating at an accelerating voltage of 120 kV using a Gatan 626 cryo-holder that maintained the specimens below -179 °C during sample transfer and observation. Images were recorded digitally on a cooled Gatan UltraScan 2k × 2k CCD camera (Gatan, U.K.) using the DigitalMicrograph software (Gatan, U.K.) in the low-dose imaging mode to minimize beam exposure and electron-beam radiation damage.33 Dynamic Light Scattering. The soft nanoparticle size was determined using a zeta-potential/particle sizer model NICOMP 380 ZLS (PSS · NICOMP, Malvern Instruments, Santa Barbara, CA). Each sample was tested three times at 25 °C. Size distributions (by volume) were obtained using the NICOMP algorithm. Light Microscopy. A small drop of each sample was placed between two glass microscope slides and observed with a Nikon light microscope Eclipse i-Series, 80i model (Tokyo, Japan) equipped with cross-polarizers attached to a Nikon digital camera DXM 1200C. Samples were examined at room temperature.
Results and Discussion A. Effect of Hydrophobic Guest Molecules on Bulk Mesophase Structure. The first set of experiments was designed to determine the effect of the added guest molecule;CBZ, CoQ10, PS, or Chl;on the structure of the GMO mesophase. All these bioactive compounds are hydrophobic, effectively solubilized at the lipid palisade layer. However, their different molecular structures and specific interaction with the lipid come into play and affect the phase behavior and properties. We examined the thermal behavior upon entrapping low equimolar quantities of each molecule (0.01 mmol of guest molecule, 0.01 mol %) below the maximum loading capacity, and at maximum loading capacity (see Table 1). We also studied the effects upon adding loads greater than the maximum encapsulation capacity. A.1. Solubilizing Equimolar Quantities of Guest Molecules in the Bulk Mesophases. QL (41:11:48 wt % of GMO/ethanol/ (32) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Microsc. Technol. 1988, 10(1), 87–111. (33) Danino, D.; Bernheim-Groswasser, A.; Talmon, Y. Colloids Surf., A: Physicochem. Eng. Aspects 2001, 183, 113–122.
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Table 2. Calorimetric Parameters for the Lβ f Lr and Lr f QL Phase Transitionsa mesophase
Lβ f LR phase transition temperature (Tm) (°C)
∆H (J/g)
∆t1/2 (°C)
LR f QL phase transition temperature (Tm) (°C)
∆H (J/g)
∆t1/2 (°C)
empty system CBZ-loaded Chl-loaded PS-loaded CoQ10-loaded
6.04 ( 0.15 5.50 ( 0.14 5.45 ( 0.15 5.69 ( 0.10 5.91 ( 0.44
-15.91 ( 0.98 -1.66 ( 0.29 -5.78 ( 0.19 -17.30 ( 0.23 -20.81 ( 0.57
3.54 ( 0.09 3.24 ( 0.12 3.40 ( 0.17 3.65 ( 0.16 3.95 ( 0.51
19.32 ( 0.42 17.51 ( 0.27 18.23 ( 0.26 18.77 ( 0.52 18.88 ( 0.54
-1.16 ( 0.12 -0.12 ( 0.10 -0.39 ( 0.14 -1.15 ( 0.20 -1.18 ( 0.23
4.16 ( 0.40 3.76 ( 0.32 3.88 ( 0.20 4.28 ( 0.30 4.69 ( 0.62
a The data was derived from the DSC curves (Figure 2A) for the empty mesophase and for the systems loaded with equimolar quantities (0.01 mmol) of each of the guest molecules.
Figure 3. Heating thermograms of the QL mesophase at maximum loading of the guest molecules.
water, 25 °C) was loaded with an equimolar quantity of each of the guest molecules (0.01 mmol, 1:217 mol ratio of solubilizate/ GMO), less than their maximum solubilization capacity. The system was cooled to -10 °C where the Lβ mesophase exists, and the effect of the solubilizates on the phase transformations taking place as a function of temperature was followed by DSC.34 These measurements also provided quantitative information on order/disorder transitions within the bilayer, and the perturbation of the lipid-lipid interactions, which indicate the localization of the guest molecules in the lipid structure. The DSC data of the empty and bioactive-loaded mesophases are shown in Figure 2A and summarized in Table 2. The thermotropic phase behavior was analyzed following the work described by Efrat et al.34 The heating scan thermograms for all the bioactive-loaded samples revealed a slightly lower transition temperature (Tm) from Lβ to LR (the main phase transition) compared with the empty system (Table 2), yet the enthalpy values of the transition event (∆H) and the peak width at half its height (∆t1/2) varied with the solubilizate type. The guest molecules CBZ and Chl significantly decreased ∆H and, to a lesser extent, ∆t1/2 of the main phase transition. ∆H of the phase transition is linked to the level at which the guest molecule affects the interactions between the lipid molecules.35,36 Chl probably orients almost parallel with the membrane surface and confers order to the lipid bilayer by inducing tighter molecular packing and restricted motion of the hydrocarbon chains.37 Similar conclusions were reached with dipalmitoylphosphatidylcholine (DPPC) bilayers, where Chl was found to roughly align along the bilayer, with the Chl hydrophobic core “buried” in the bilayer hydrocarbon region and the OH groups located close to the (34) Efrat, R.; Aserin, A.; Garti, N. Spec. Publ. - R. Soc.Chem. 2006, 308, 87–102. (35) Kostecka-Gugala, A.; Latowski, D.; Strzalka, K. Biochim. Biophys. Acta: Biomembr. 2003, 1609(2), 193–202. (36) Sujak, A.; Strzalka, K.; Gruszecki, W. I. Chem. Phys. Lipids 2007, 145(1), 1–12. (37) Kessel, A.; Ben-Tal, N.; May, S. Biophys. J. 2001, 81(2), 643–658.
chains-headgroup boundary.38,39 Sankaram and Thompson40 showed that the 3β-hydroxyl of Chl creates hydrogen bonds with sn-2-carbonyl of the DPPC. Thus, we suggest CBZ and Chl, which we classify as type I molecules, are localized within hydrophobic moieties in the vicinity of the lipid headgroups, with their stiff rings and the functional groups located near the chain-headgroup region. At this location, they influence the order of the lipid acyl chains as well as electrostatic interactions and hydrogen bonds between the lipid headgroups. Trouard et al.41 demonstrated that solubilized Chl in dimyristoylphosphatidylcholine (DMPC) bilayers decreased the number of rotametric degrees of freedom and the chain entanglements, and increased the ordering at the upper part of the acyl groups (near the polar group). Thus, the lower ∆t1/2 values we find with type I-loaded systems (compared with the empty system) likely indicate increased rigidity of the interface in the presence of Chl or CBZ. Thus, we conclude that perturbation of the lipid bilayer by type I molecules is large, resulting in significant reduction in ∆H compared with the empty system. CBZ effect on ∆H is more pronounced than that of Chl, reflecting the difference in their molecular structure. The bulky CBZ (composed of only a tricyclic ring) imposes a large steric hindrance on the headgroups and hence largely influences ∆H.42 On the other hand, as discussed, the planar Chl likely orients almost parallel with the membrane surface,37 and therefore possess a smaller steric effect. In addition, CBZ solubility in water is an order of magnitude greater than that of Chl (170 and 11.95 µg/ mL for CBZ and Chl, respectively),25,42-45 suggesting CBZ molecules may be located closer than Chl to the headgroup region. Thus, we conclude that CBZ molecules disturb mesophase packing, especially at the headgroup region, more than Chl, thereby imparting a stronger effect on the thermal parameters. The two other solubilizates, PS and CoQ10, which we classified as type II molecules, exhibit opposite and relatively moderate effects on the thermal parameters. The phase transition enthalpies of PS and CoQ10 are slightly greater than that of the empty system (Table 2), while the peak heights of the empty and loaded systems are similar (Figure 2A). However, the widths of the peaks (∆t1/2) of the loaded systems are wider than those of the empty QL mesophase (Table 2). The slight broadening of the peak in the presence of PS or CoQ10 suggests they may act as small impurities. The larger effect of CoQ10 on ∆H and ∆t1/2 relative to that of PS may be linked with differences in their tail (38) Leonard, A.; Escrive, C.; Laguerre, M.; Pebay-Peyroula, E.; Neri, W.; Pott, T.; Katsaras, J.; Dufourc, E. J. Langmuir 2001, 17(6), 2019–2030. (39) Villalain, J. Eur. J. Biochem. 1996, 241(2), 586–593. (40) Sankaram, M. B.; Thompson, T. E. Proc. Natl. Acad. Sci. U.S.A. 1991, 88(19), 8686–8690. (41) Trouard, T. P.; Nevzorov, A. A.; Alam, T. M.; Job, C.; Zajicek, J.; Brown, M. F. J. Chem. Phys. 1999, 110(17), 8802–8818. (42) BenShaul, A.; Ben-Tal, N.; Honig, B. Biophys. J. 1996, 71(1), 130–137. (43) Fourie, L.; Breytenbach, J. C.; Du Plessis, J.; Goosen, C.; Swart, H.; Hadgraft, J. Int. J. Pharm. 2004, 279(1-2), 59–66. (44) Jorgensen, K.; Ipsen, J. H.; Mouritsen, O. G.; Bennett, D.; Zuckermann, M. J. Biochim. Biophys. Acta 1991, 1067(2), 241–253. (45) Matsuoka, K.; Kuranaga, Y.; Moroi, Y. Biochim. Biophys. Acta: Mol. Cell Biol. Lipids 2002, 1580(2-3), 200–214.
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Table 3. Calorimetric Parameters for the Lβ f Lr and Lr f QL Phase Transitionsa mesophase
Lβ f LR phase transition temperature (Tm) (°C)
∆H (J/g)
∆t1/2 (°C)
LR f QL phase transition temperature (Tm) (°C)
∆H (J/g)
∆t1/2 (°C)
empty system QL+CoQ10 QL+PS
6.04 ( 0.15 5.20 ( 0.13 5.56 ( 0.17
-15.91 ( 0.98 -6.96 ( 0.48 -0.35 ( 0.61
3.54 ( 0.09 3.42 ( 0.11 3.03 ( 0.14
19.32 ( 0.42 18.22 ( 0.25 17.31 ( 0.31
-1.16 ( 0.12 -0.73 ( 0.14 -0.06 ( 0.10
4.16 ( 0.40 3.98 ( 0.35 3.19 ( 0.39
a
The data was derived from the DSC curves for the empty mesophase and systems with a maximum load of type II guest molecules.
Figure 4. Characteristic birefringent texture under crossed polarizers of QL loaded with guest molecules above the maximum solubilization capacity. (A,B) Samples containing CBZ, showing crystals (A) and lamellar texture in the form of an oily streak network (B). Chl solutions exhibit characteristic Chl crystals (C) and lamellar phase + giant multilamellar vesicles (D and inset). (E) PS crystals; (F) CoQ10 crystals. Bars ) 100 µm in B and D, and 25 µm in all other panels.
length: that of CoQ10 is 2.5 times longer than that of GMO (ca. 5.0 vs 2.1 nm at room temperature), while for PS it is similar to that of GMO (ca. 1.9 nm).40,46,47 The locus of CoQ10 within the lipidic bilayer intrigued several researchers.27,48,49 Three locations were proposed: (1) near the water-lipid interface, (2) in the hydrophobic acyl chain area of the membrane (far from the lipid-water interface), or (3) sandwiched between the two monolayers composing the phospholipid bilayer. The small effects of type II molecules on Tm, (46) Pata, V.; Dan, N. Biophys. J. 2005, 88(2), 916–924. (47) Di Bernardo, S.; Fato, R.; Casadio, R.; Fariselli, P.; Lenaz, G. FEBS Lett. 1998, 426(1), 77–80. (48) Afri, M.; Ehrenberg, B.; Talmon, Y.; Schmidt, J.; Cohen, Y.; Frimer, A. A. Chem. Phys. Lipids 2004, 131(1), 107–121. (49) Gomez-Fernandez, J. C.; Llamas, M. A.; Aranda, F. J. Eur. J. Biochem. 1999, 259(3), 739–746.
∆H, and ∆t1/2 suggest that the quinone rings of CoQ10 are located either in the hydrophobic acyl chain area of the bilayer, or possibly sandwiched between the two monolayers. Analysis of our data at maximum load (discussed below) better supports model 2, namely localization of type II molecules mainly within the lipid chains. The guest molecules’ effect can be further explored by examining their influence on the second phase transition, from LR to QL. The general behavior was similar to that exhibited in the main phase transition, with the two types of solubilized molecules inducing different effects on ∆H and ∆t1/2. Additionally, as in the Lβ-to-LR transition, all loaded samples exhibited slightly lower Tm values compared with the empty system (Table 2).
Effect of Guest Molecules on Lipid Cubic Structures
Figure 5. SAXS diffraction patterns exhibiting two Bragg peaks in the positional ratio of 1:2, in QL solutions loaded with 1.5 wt % CBZ (A) and 9 wt % Chl (B), above the maximum loading capacity.
Figure 6. (A) Cubosomes form upon dispersion of the QL mesophase; they coexist with vesicles. The internal long-range cubic order is clearly visible at higher magnification (B), and is further confirmed by the Fourier transformation given in the inset. (C) SAXS diffraction patterns showing the differences in intensity and position of peaks between the bulk QL mesophase and the dispersed nanoparticles. (D) Particle size distribution immediately after preparation (gray line) and after incubation for 5 months (black line).
PS and Chl are structurally related, yet their thermal behavior is remarkably different, clearly demonstrating that the lipid-guest compound interactions are strongly dependent on the fine structure of the guest molecules. PS components contain an additional short carbon chain attached at the C-24 position,31,34 imparting more lipophilic characteristics to PS over Chl. Similar data were reported by Halling and Slotte,50 showing that plant sterols such as campesterol, β-sitosterol, and stigmasterol do not interact as favorably as Chl with D-erythro-N-palmitoyl-sphingomyelin (PSM). The data presented here show that the thermal behavior, in the presence of even small amounts of hydrophobic guest molecules, can reflect the influence of these molecules on the lipid structure and their location in the bilayer. We also found that molecular characteristics (structure, geometrical constraints) dictate the localization of the guest molecules, with the main changes in the thermograms resulting from perturbation of the interactions near the lipid headgroups. SAXS analysis (Figure 2B) showed that all the loaded systems exhibited (at this low loading) the same peak ratios as the empty (50) Halling, K. K.; Slotte, J. P. Biochim. Biophys. Acta: Biomembr. 2004, 1664(2), 161–171.
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system (2; 6; 8; 12, indexed as a primitive space group P4232, P4332, or Pm3n). Yet, higher intensities were measured for the systems loaded with type II solubilizates (CoQ10 and PS) compared with the systems loaded with type I molecules (Chl and CBZ), in support of the differences in the thermal behavior these two types of molecules present. A.2. Maximum Solubilization of the Guest Molecules. Samples containing maximum solubilization quantities remained liquid, optically isotropic, and completely transparent without any indication of crystal deposition (detected by polarized light microscopy), meaning that the guest molecules were fully molecularly entrapped within the mesophase. The maximum solubilization levels of the four guest molecules we studied are shown in Table 1. Only low levels of CoQ10 and CBZ could be efficiently encapsulated, likely due to the strong perturbation caused by their long hydrophobic tail (CoQ10) or bulky structure (CBZ).51 The sterol loading, on the other hand, is relatively high, probably reflecting good matching of the hydrophobic domain to that of the lipid (∼20 Å in length for both GMO and the sterols). Yet, the Chl maximum encapsulation capacity is twice as great as that of PS, possibly because the additional side-chain of PS perturbs the planar structure and induces a steric hindrance. Similar effects were reported for Chl versus lanosterol in phospholipid bilayers.52 The thermal behavior of the guest molecules at their maximum loading capacity follows the tendency identified at the low encapsulation levels. For type I solubilizates (CBZ, Chl), the main phase transitions (Lβ f LR and LR f QL) were significantly reduced, and they could not be detected (Figure 3). Apparently, perturbations of the headgroup interactions by these molecules and the resulting decrease in the internal mesophase order and cohesion forces significantly decreased the phase transition enthalpy. Early investigations of DPPC bilayers revealed a similar phenomenon, i.e., the total area of the main endothermic peak of the transition was undetectable at high Chl loads.52 Type II solubilizates (PS, CoQ10) also decreased ∆H (as well as Tm and ∆t1/2 values; see Table 3) but to a lesser degree, in line with the presumed location of type II molecules far from the headgroups, and their smaller perturbation of the lipid molecules. Thus, as found at the low solubilization levels (Table 2), the two types of guest molecules perturb the lipid interactions at maximum loading; however, type I molecule perturbation is larger, thus more strongly affecting the thermal characteristics. Because of the lower degree of internal order, SAXS diffractions of all guest molecules at the maximum solubilization capacity are rather diffused and display a strong loss of resolution compared with the empty system (figure not shown). Therefore, the space groups of these loaded mesophases could not be determined. A.3. Structural Transitions in Bulk Mesophases aboVe Maximum Solubilization Capacity. Adding type I molecules to QL above the maximum solubilization capacity induced phase separation at room temperature into QL and lamellar phases. This was detected by polarized light microscopy and SAXS. The transition into the lamellar mesophase can be explained by changes in the bilayer curvature upon solubilization of these guest molecules close to the headgroup region, in line with the conclusion from the DSC data discussed above. In CBZ solutions, only a thin layer of a lamellar phase formed, but after storage for 2 days at room temperature, in the presence of concentrations greater than 1.5 wt % CBZ, needle-like CBZ crystallites were detected (Figure 4A). The lamellar phase exhibited an oily streak network of different sizes (Figure 4B)
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Figure 7. (A) cryo-TEM image of the dispersed Chl-loaded system showing two populations of unilamellar vesicles. Most vesicles are 25-45 nm in diameter; a small population is in the range of 180-210 nm. (B,C) dispersion of the empty QL system. (D,E) Dispersed nanoparticles made from the lamellar GMO-EtOH-water mesophase showing unilamellar and multilamellar vesicles. (F) Cryo-TEM image of the dispersed CBZ-loaded system showing vesicles, intermediate structures with some internal order, and sponge-like structures. (G) Three-dimensional representation of the sponge-like surface of a typical nanoparticle. (H,I) Interlamellar attachment (ILA) arrays at different degrees of development of the nanoparticles. Inset in (F) is a Fourier transformation showing the lack of long-range order within the nanoparticles. White arrows in (F) point to small unilamellar vesicles (1), intermediate structures with some internal order (2 and 3), and ILA structures (4).
described as aggregates of focal conic domains.53 SAXS diffraction patterns of the lamellar (upper) phase revealed two Bragg peaks in the positional ratio of 1:2 and a repeating distance of 50.63 Å (Figure 5A), consistent with the existence of a lamellar phase. The overloaded Chl solutions show similar phase separation into lamellar and QL mesophases, with inverted proportions. The fraction of the lamellar phase increased with Chl content up to 9 wt %, then Chl precipitated as platelet crystals (Figure 4C). The SAXS diffraction curves of the stable solution revealed two typical lamellar Bragg peaks at a 1:2 ratio, with a repeating distance of 54.61 Å (Figure 5B). Polarized light microscopy of the lamellar phase showed giant multilamellar vesicles (>20 µm in diameter, Figure 4D and inset) rather than a classical lamellar pattern. Thus, it seems that Chl increases the bilayer curvature, thereby enhancing formation of the lamellar phase. At the same (51) Rodriguez-Hornedo, N.; Murphy, D. J. Pharm. Sci. 2004, 93(2), 449– 460. (52) Mannock, D. A.; Lewis, R. N. A. H.; McElhaney, R. N. Biophys. J. 2006, 91(9), 3327–3340. (53) Boltenhagen, P.; Lavrentovich, O.; Kleman, M. J. Phys. II 1991, 1(10), 1233–1252.
time it increases the bilayer elasticity, enabling bending of the lamellar mesophase and vesicle formation.54 In samples containing type II molecules in quantities greater than the maximum solubilization capacity, the solubilizates crystallized (Figure 4E, F) within a couple of days, without a preceding noticeable phase separation. As discussed earlier, CoQ10 and PS molecules are localized at the hydrophobic domain, far from the tail-headgroup interface. At this location, they cannot alter the lipid curvature or transform the QL mesophase into another phase; therefore, above maximum loading, an excess of solubilizate precipitates in crystal form. B. Characterization of Dispersed Soft Nanoparticles of the QL Mesophase. Dispersions of the QL mesophase were prepared with 41 wt % GMO, 11 wt % EtOH, and 48 wt % water using F-127, in a 1:10 weight ratio of F-127 to QL. At this ratio, F-127 was sufficient to facilitate the formation of stable nanoparticles. Starting with the cubic-micellar QL mesophase, only low shear was required for the dispersion, as also found by Spicer and co-workers when diluting the micellar GMOEtOH-water phase (without passing through the QL region) or (54) Gradzielski, M. J. Phys.: Condens. Matter 2003, 15(19), R655–R697.
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Figure 8. SAXS diffraction patterns of the bulk mesophases loaded with solubilizates, and the corresponding dispersed systems: (A) Chl-loaded systems, (B) CBZ-loaded systems, (C) PS-loaded systems, (D) CoQ10-loaded systems.
the emulsion region.19 The homogenized dispersions were investigated by cryo-TEM, SAXS, and particle size analysis. Cryo-TEM images revealed the formation of soft nanoparticles with an internal cubic order (cubosomes), which, as in other GMO-based dispersions,17,55 coexist with a relatively large fraction of small and medium vesicles (Figure 6A). The cubosome sides range from ∼200 to ∼500 nm. Fourier analysis indicated a periodicity of 97 Å (Figure 6B), which is smaller than the lattice parameter of 108 Å we determined for the QL mesophase.23 Differences between the cubosomes and the QL mesophase structures were also identified in the SAXS scattering curves (Figure 6C). In general, the intensity of the peaks of such dispersed nanoparticles is weak.21 The three Bragg peaks that were resolved in the SAXS curves were consistent with the presence of cubic symmetry (Figure 6C). Their relative positions in ratios 2, 4, and 6 could match a body-centered cubic phase of space group Im3m (Q229) or primitive cubic Pn3m or Pm3n, assuming some peaks are missing. The lattice parameter was calculated to be 99.7 Å (R ) 0.999), in good agreement with the lattice parameter evaluated from the cryo-TEM data. For the QL, as shown in Figure 6C, the analysis could fit space groups Pm3n, P4232 or P4332.23 The particle sizes were estimated from DLS measurements, immediately after preparation and after incubation for 5 months. In the samples analyzed soon after preparation, three main populations were detected, with mean diameters of 19, 73, and (55) Gustafsson, J.; Ljusberg Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12(20), 4611–4613.
248 nm, and a polydispersity index of about 0.315 (Figure 6D, gray line). From the cryo-TEM data we learn that the small and medium-size populations are vesicles, while the large particles are cubosomes. After 5 months, DLS revealed only two main populations with mean diameters of 97 and 333 nm, and a polydispersity index of about 0.367. Thus we find the smallest population disappears over time and the other two populations slightly increase. This can be explained by fusion events between nanoparticles. This possibility is discussed further in the following sections. B.1. Shape and Structure of Dispersed Nanoparticles of the Loaded Systems. Nanoparticles were prepared from the QL systems loaded at their maximum capacity (Table 1), and the effect of the guest molecules on the internal microstructure of the soft nanoparticles was studied by cryo-TEM and SAXS. As for the bulk mesophases, we could group the solubilizates into type I and type II molecules, based on the internal microstructure of the soft nanoparticles. B.1.1. Type I Molecules. Upon dispersion of the Chl-loaded system, the internal order was completely destroyed and only spherical vesicles formed, with two characteristic populations, 25-45 and 180-210 nm in diameter (Figure 7A). This result is in line with the effects found for the bulk system where adding high Chl loads to QL transforms it into lamellar phase and vesicles (Figures 4 and 5). As discussed above, vesicles were also created upon dispersing the empty QL mesophase (at the same composition and procedure); however those vesicles coexisted with cubosomes, and part of them were oligo-vesicular (Figure 7B, C), while, in
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Figure 9. (A,B) cryo-TEM image of the dispersed PS-loaded system showing small unilamellar vesicles (∼30 nm in diameter), complex intermediate structures, and soft nanoparticles with defined cubic symmetry. (C) Cryo-TEM image of the dispersed CoQ10-loaded system showing vesicles, less ordered particles, and well-ordered square-shaped nanoparticles with internal cubic order. The Fourier transformation shown in the inset further supports the internal cubic order of the particles. (D,E) 3D-imaging presentation of the cubic surface of a typical nanoparticle.
the presence of Chl, only unilamellar vesicles are noted. To confirm that lack of fusion between nanoparticles or formation of ordered nanostructures is due to the presence of Chl, we dispersed the lamellar GMO/EtOH/water mesophase (wt ratio 70:10:20). This resulted in the formation of a mixture of small unilamellar vesicles, and larger bi- or multilamellar vesicles (Figure 7D, E). Thus, the single system where only unilamellar vesicles were formed upon dispersion was that containing Chl, supporting our conclusion that Chl inhibits the formation of complex nanoparticles. Our results are in line with studies by Yeagle et al.56 and Wenk and Seeling,57 reporting that addition of lysophospatidylcholine (LPC) to the lipid N-methylated (56) Yeagle, P. L.; Smith, F. T.; Young, J. E.; Flanagan, T. D. Biochemistry 1994, 33(7), 1820–1827. (57) Wenk, M. R.; Seelig, J. Biochim. Biophys. Acta: Biomembranes 1998, 1372(2), 227–236.
dioleoylphosphatidylethanolamine (DOPE) inhibits fusion of large unilamellar vesicles (LUVs). No indication of ordered domains or periodicity were found by SAXS (Figure 8A), in support of the cryo-TEM data. Indeed, similar scattering curves were interpreted as characteristic patterns of unilamellar vesicles.58 The system containing 0.35 wt % CBZ (maximum loading) was dispersed by the same procedure. Here, quite different dispersed particles were formed. Cryo-TEM images revealed the existence of vesicles (Figure 7F, arrow 1) and intermediate structures with some internal order (Figure 7F, arrow 2). The vesicles are small (around 25 nm in diameter), and they are unilamellar. The intermediate nanoparticles are at different transition degrees between vesicles and a dense unordered (58) Sagalowicz, L.; Mezzenga, R.; Leser, M. E. Curr. Opin. Colloid Interface Sci. 2006, 11(4), 224–229.
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Figure 10. Suggested path of the growth process of cubosomes, starting from unilamellar vesicles. Initial stages of fusion events between unilamellar vesicles with spherical particles are shown in line 1. Advanced stages of fusion are created by many ILAs between opposed bilayers (line 2), eventually leading to the creation of aggregates with cubic internal order and a square shape (line 3). Table 4. Size Distribution of the Nanoparticles As Measured by DLS NICOMP volume-weighted mean value (nm)
volume (%)
dispersion composition
polydispersity index
population I
population II
population I
population II
Chl CBZ PS CoQ10
0.082 0.141 0.240 0.108
38.2 143.5 40.9 143.9
205.3 298.3 188.5 270.9
4.0 39.4 79.5 40.8
96.0 60.6 20.5 59.2
structure, which may be sponge-like (L3) structures (Figure 7F, arrow 3). Fusions between elements are clearly visible in these nanoparticles by identification of the ILA arrays (Figure 7H, I). The sponge phase can be described as a melted form of the bicontinuous cubic mesophase since for both of these phases the infinite membrane bilayer presents saddle-like topology with local principal curvatures of opposite signs. However, in contrast with the cubic mesophases, L3 does not have long-range order and periodicity.59,60 Indeed, further analysis of the images by Fourier transformation showed no periodicity, consistent with the lack of long-range order to be expected from L3 phases (Figure 7F, inset). Applying the 3D-imaging software to these images we could better stress the existence of disordered surfaces of the lipid within these nanoparticles (Figure 7G). While the inner interface of the particles is unordered, the outer area of the sponge-like phase is indicative of ILA structures (Figure 7F, arrow 4), similar to those reported by Rangelov and Johnsson.61,62 The lack of any cubic order indicates that, under the conditions we examined, the ILAs will likely not proceed to create as ordered a phase as the cubic one. Previous reports show that ILAs are precursors of the bicontinuous cubic mesophase.63-67 Our data suggest they can also be precursors of (59) Porcar, L.; Hamilton, W. A.; Butler, P. D. Langmuir 2003, 19(26), 10779– 10794. (60) Zapf, A.; Hornfeck, U.; Platz, G.; Hoffmann, H. Langmuir 2001, 17(20), 6113–6118. (61) Johnsson, M.; Edwards, K. Biophys. J. 2001, 80(1), 313–323. (62) Rangelov, S.; Almgren, M. J. Phys. Chem. B 2005, 109(9), 3921–3929. (63) Kozlovsky, Y.; Efrat, A.; Siegel, D. A.; Kozlov, M. M. Biophys. J. 2004, 87(4), 2508–2521.
the sponge phase. Moreover, the present data strengthen our previous conclusion that, like Chl, CBZ affects the spontaneous curvature, likely by localizing close to the headgroup region. The SAXS diffraction curve of the dispersed CBZ-loaded system exhibited a wide peak (Figure 8B), which is typical of a sponge phase,68,69 in support of the cryo-TEM findings. B.1.2. Type II Molecules. Unlike type I molecules, solubilizates of type II did yield soft nanoparticles with cubic symmetry. The dispersed PS-loaded system consisted of small unilamellar vesicles (∼30 nm in diameter), complex intermediate structures, and soft nanoparticles with defined cubic symmetry (Figure 9A). The core of these intermediate nanoparticles displayed regions with varying degrees of order and longer periodicity (Figure 9B). A series of intermediate structures, showing possible stages of development from vesicles to ordered nanoparticles with cubic arrangement by fusion of the vesicular elements via ILA structures is presented in Figure 10. The process begins by fusion events between unilamellar vesicles with spherical particles (Figure 10, line 1). At advanced stages, “irregular globules” with many connections between opposed bilayers are created, forming an array that could allow the formation of structures with long(64) Siegel, D. P. Biophys. J. 1986, 49(6), 1171–1183. (65) Siegel, D. P. Biophys. J. 1999, 76(1), 291–313. (66) Siegel, D. P. Surfactant Sci. Ser. 2005, 27, 59–98. (67) Gotter, M.; Strey, R.; Olsson, U.; Wennerstrom, H. Faraday Discuss. 2005, 129, 327–338. (68) Bender, J.; Jarvoll, P.; Nyden, M.; Engstrom, S. J. Colloid Interface Sci. 2008, 317(2), 577–584. (69) Gomati, R.; Bouguerra, N.; Gharbi, A. Physica B 2001, 299(1-2), 101– 107.
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range order and cubic symmetry (Figure 10, line 2). We consider this a precursor of the cubic structure. As more fusions take place, structure complexity increases, eventually leading to the creation of aggregates with cubic order exhibiting square or almost square shape (Figure 10, line 3). This mechanism is in accordance with the modified stalk theory suggested by Siegel, of membrane fusion and formation of inverted phases through a large number of ILAs.64,65 Thus, in contrast to the results with CBZ, cubic assembly inside nanoparticles can build up with PS as the guest molecule.65,66,70,71 The formation of these nanoparticles with cubic order is in line with the data we reported above for the bulk mesophase, where PS was either incorporated within the cubic mesophase while preserving its internal order, or precipitated out as crystals above the maximum encapsulation capacity without interfering with the internal order. X-ray diffraction of the PS-loaded dispersions reveals the existence of four Bragg peaks at relative positions of 2, 3, 6, and 9. This is a characteristic pattern of cubic P4332, Pn3m, or less probably of P4232, assuming some peaks are missing. This periodicity rules out space group Im3m, which was one of options we found for the unloaded nanoparticles. The lattice parameter, calculated from the Fourier transformation, was found to be 87.2 Å (Figure 9B, inset). Dispersion of QL loaded with the second solubilized molecule of type II, CoQ10 (0.5 wt%), results in the formation of mostly well-ordered square-shaped nanoparticles with internal cubic order, as shown in Figure 9C and the inset. Some particles had a spherical shape and displayed lower internal order. Unilamellar vesicles (mainly small, 31-47 nm) also existed. The 3D images of the cubic nanoparticles created from the cryo-TEM by ImageJ (Figure 9D, E) are consistent with long-range order and cubic symmetry. Four Bragg diffraction peaks were detected in the X-ray diffractograms of these nanoparticles. The peaks were spaced in ratios of 2:3:6:9, and could be indexed as hkl: (110), (111), (211), and (221) reflections, in agreement with the primitive cubic lattice Pn3m assuming the (200) peak (4) is missing as a result of the low intensity (Figure 8D). This is the same space group we found for the PS-loaded nanoparticles. A lattice parameter of 91.8 Å was found, somewhat smaller than that calculated for the dispersed soft cubic nanoparticles of the empty QL, and closer to the values found for the other loaded systems. In summary, it is shown that the presence of type II solubilizates, which reside relatively far from the lipid headgroup area, enables the formation of nanoparticles with cubic symmetry. B.2. Nanoparticle Size Distribution. The “NICOMP distribution model” revealed two populations for all samples (Table 4), with the vesicles constituting the smaller-size population (unilamellar or few fused vesicles, as detected by cryo-TEM) and the large particles constituting the structured nanoparticles, except for Chl, where only unilamellar vesicles existed. The polydispersity index of the Chl-containing system was found to be relatively small, indicating the presence of a rather homogeneous population of nanoparticles.
Conclusions Four lipophilic biomolecules, CBZ, CoQ10, PS, and Chl, were solubilized in the discontinuous QL mesophase of monoolein. (70) Knecht, V.; Marrink, S. J. Biophys. J. 2007, 92(12), 4254–4261. (71) Siegel, D. P.; Burns, J. L.; Chestnut, M. H.; Talmon, Y. Biophys. J. 1989, 56(1), 161–169.
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Analysis of their effects on the enthalpy changes in the presence of even small quantities of guest molecules permitted their sorting into two types, and provided insight into their localization in the bilayer and their influence on the phase behavior (e.g., phase transitions, phase separation) at high loads. Our experiments suggest type I molecules (CBZ and Chl) are probably localized within the plaside of the lipid with their hydrophilic moieties arranged in the vicinity of the lipid headgroups, while type II molecules (PS and CoQ10) appear to be located mainly within the lipid tails, relatively far from the hydrophilic lipid moiety. The effect of guest molecules on the formation and structure of nanoparticles upon dispersion of the drug-loaded mesophase is consistent with their influence on the bulk mesophase. Type I solubilizates affect the lipid curvature, thus yielding soft nanoparticles with no or low internal order. Type II molecules do not change the packing, thus they allow the formation of cubosomes. We recognized that the nanoparticles are reconstructed from the solution by a bottom-up process through fusion events between lipid elements. The growth starts by fusion between two small vesicles to form intermediate structures. As the process proceeds through additional fusions, more complex intermediates form with bigger ordered domains, up to the formation of a cubic array. The soubilizate type dictates the final particulate structure and determines the degree of the internal order: the smaller the effect on the Gaussian curvature and the elastic modulus of the lipid monolayers, and on the lipid-lipid interactions, the more developed the order inside the nanoparticles. It was interesting to note that the solubility capacity of the monoolein discontinuous QL mesophase was very much dependent on the molecular characteristics of the guest molecules, and independent of their localization within the lipid bilayer. Thus, much larger loads of the flat Chl and PS molecules could be incorporated in the lipid compared with the quantities of the bulky CBZ and long CoQ10. In line with this result, it is further shown that the solubility of PS, although large, is less than that of Chl, due to the steric hindrance caused by its additional side chain at the C-24 position. The presence of this side chain also causes PS to localize to the more hydrophobic domains of the lipid compared with Chl. Acknowledgment. We acknowledge the generous support of the United States-Israel Binational Science Foundation, Grant Number 2003260. The cryo-TEM work was performed at the Cryo-TEM Hannah and George Krumholz Laboratory for Advanced Microscopy, at the Technion. D.D. acknowledges the support of the Matilda Barnett Recoverable Trust and the Israel Science Foundation of the Israel Academy of Sciences and Humanities (Grant No. 9059/03), and the support of RBNI. Part of the results presented in this paper have been included in the dissertation of R.E. for the degree of Doctor in Applied Chemistry at The Hebrew University of Jerusalem, Israel. Note Added after ASAP Publication. This article was released ASAP on September 10, 2008. A change was made to the caption of Figure 9C, and the article was reposted on January 27, 2009. LA8016084
