Letter pubs.acs.org/Langmuir
Generation of Geometrically Ordered Lipid-Based Liquid-Crystalline Nanoparticles Using Biologically Relevant Enzymatic Processing Wye Khay Fong,† Stefan Salentinig,† Clive A. Prestidge,‡ Raffaele Mezzenga,§ Adrian Hawley,∥ and Ben J. Boyd*,† †
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia ‡ Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095, Australia § ETH Zürich, Food & Soft Materials Science, Department of Health Science & Technology, Schmelzbergstrasse 9, LFO E23, 8092 Zürich, Switzerland ∥ SAXS/WAXS Beamline, Australian Synchrotron, 800 Blackburn Road, Clayton, VIC 3168, Australia S Supporting Information *
ABSTRACT: High-symmetry lipid nanoparticles with internal bicontinuous cubic phase structure (cubosomes) are prepared from a simple emulsion containing a mixture of a nondigestible lipid (phytantriol) and a digestible short-chained triglyceride using enzymatic lipolysis of the incorporated short-chained triglyceride. The lipolytic products partition away from the nondigestible lipid, resulting in crystallization of the cubic-phase internal structure. Timeresolved small-angle X-ray scattering revealed the kinetics of the disorder-to-order transition, with cryo-transmission electron microscopy showing an absence of liposomes. The new approach offers a new “sideways” method for the generation of lipid-based nanostructured materials that avoids the problems of top-down and bottomup approaches.
■
action of lipolytic enzymes such as pancreatin.17 It is unequivocal that such structures are formed at the surface of a digesting lipid droplet, and it is likely that they detach from the surface as structured particles with a hexagonal and/or cubic internal phase. The generation of liquid-crystalline particles during in vitro digestion has been shown under highly controlled pH conditions during the digestion of milk;18 however, the particles have also been shown to be destroyed during digestion when constructed from digestible lipids such as glyceryl monooleate19 but not when prepared with nondigestible lipids such as phytantriol (PHYT).20 A novel approach to creating these internally ordered particles is proposed in this Letter, illustrated schematically in Figure 1, which utilizes the biological processing of lipids during digestion to generate internal nanostructure which persists on completion of the digestion process. Specifically, precursor disordered emulsion systems were prepared by the dispersion of a mixture of digestible and nondigestible lipid components which, under the action of lipase enzyme, undergo a transformation to particles possessing the highly ordered bicontinuous cubic phase. The process hinges on (i) the selection of an appropriate digestible lipid that disorders the
INTRODUCTION Lipid-based self-assembled nanomaterials with high geometric order, such as the inverse bicontinuous (V2) and reversed hexagonal (H2) phases, have great potential for applications in drug delivery,1,2 food science,3 biosensing,4,5 and protein crystallization.6,7 These self-assembled nanomaterials are used to encapsulate and protect a wide range of guest molecules and control their release from within the tortuous nanostructure of the liquid-crystalline phase. However, the high viscosity of these mesophases often limits their use.8 As these matrices are thermodynamically stable, they can be dispersed into colloidal particles while retaining the internal order of the bulk material, thus overcoming this limitation but necessitating the use of a colloidal stabilizer.9 Such particles with internal bicontinuous cubic phase structure have been termed cubosomes.10 The preparation of dispersed liquid-crystalline particles typically requires high-intensity sonication,11,12 high shear,13 or highpressure homogenization14 in top-down approaches or the incorporation of hydrotropes to form particles on dilution15,16 in a bottom-up approach. However, the top-down approaches are energy-intensive and are not desirable when incorporating sensitive elements such as drugs and proteins, while controlling the particle size distribution with the dilution approaches is practically challenging. Nature is well known to transiently produce hierarchically structured materials in vivo during lipid digestion under the © 2014 American Chemical Society
Received: February 4, 2014 Revised: April 30, 2014 Published: May 1, 2014 5373
dx.doi.org/10.1021/la5003447 | Langmuir 2014, 30, 5373−5377
Langmuir
Letter
Figure 1. Left panel: Schematic illustrating the lipolysis-induced transformation from a disordered emulsion or dispersed L2 phase system to cubosomes as a consequence of the digestion of triglyceride and the release of digestion products to the aqueous medium, leaving the water-insoluble phytantriol to form cubosomes as it would in excess aqueous solution. Right panel: Molecular structures of phytantriol and the incorporated shortchain triglycerides. in 1 mL of digestion buffer (Tris maleate 50 mM, 150 mM NaCl, 5 mM CaCl2·2H2O, pH 7.5) was added to a mixture of 1 mL of an L2 phase dispersion and 8 mL of digestion buffer25 using a remotely driven syringe pump, while the digesting medium was pumped through a 1.5 mm quartz capillary flow cell at 10 mL/min using a peristaltic pump.20 The quartz capillary was placed in the X-ray beam at the SAXS/WAXS beamline at the Australian Synchrotron.26 The scattering profiles were acquired for 5 s every 10 s at an energy of 10 keV using a Pilatus 1 M detector (active area 169 × 179 mm2 with a pixel size of 172 μm) with a sample-to-detector distance of 1015 mm providing a q range of 0.01 < q < 0.7 Å−1, where q is the length of the scattering vector defined by q = 4π/λ sin(θ/2), with λ being the wavelength and θ being the scattering angle. The scattering images were integrated into the one-dimensional scattering function I(q) using the in-house-developed software package ScatterBrain. The qrange calibration was made using silver behenate as the standard. The cubic- and hexagonal-phase space groups and lattice parameters were determined by the relative positions of the Bragg peaks in the scattering curves, which correspond to the reflections on planes defined by their (hkl) Miller indices.27 For the equilibrium phase studies, dispersions were added to a 96-well plate, and scattering was acquired for 1 s as previously described.24 For cryo-TEM studies, a laboratory-built humidity-controlled vitrification system was used to prepare the samples for cryo-TEM. Humidity was kept close to 80% for all experiments, and the ambient temperature was 22 °C. Copper grids (200 mesh) coated with a perforated carbon film (lacey carbon film: ProSciTech, Qld, Australia) were glow discharged in nitrogen to render them hydrophilic. Aliquots (4 mL) of the sample were pipetted onto each grid prior to plunging. After 30 s of adsorption time, the grid was blotted manually using Whatman 541 filter paper for approximately 2 s. The blotting time was optimized for each sample. The grid was then plunged into liquid ethane cooled by liquid nitrogen. Frozen grids were stored in liquid nitrogen until required. The samples were examined using a Gatan 626 cryoholder (Gatan, Pleasanton, CA, USA) and a Tecnai 12 transmission electron microscope (FEI, Eindhoven, The Netherlands) at an operating voltage of 120 kV. The sample holder operated at −175.5 ± 1 °C. At all times, low-dose procedures were followed, using an electron dose of 8−10 electrons/Å2 for all imaging. Images were recorded using an FEI Eagle 4kx4k CCD camera at magnifications ranging from 15 000× to 50 000×.
cubic phase to an unstructured emulsion prior to digestion for easy dispersion and (ii) the elimination of digestion products from the droplet through partitioning to allow the crystallization of the geometric internal structure. Short-chain triglycerides, when mixed with phytantriol, were hypothesized to provide this set of properties when converted from the triglyceride to the short-chain fatty acids and glycerol. To test this hypothesis, the effect of the addition of triacetin (TA), tripropionin (TP), and tributyrin (TB) (Figure 1) on the equilibrium internal structure of phytantriol cubosomes was first established to determine the phase boundaries where disorder was induced. With the advent of advanced synchrotron methods, we are now able to kinetically resolve structure formation during fast processes in soft matter systems, such as the responses of a structure to a wide range of stimuli,21,22 including the digestion of lipid-based self-assembled systems, on time scales shorter than minutes using small-angle X-ray scattering (SAXS).20 Hence, time-resolved SAXS was then used in concert with a flow cell fitted to a lipolysis model as reported previously20 to enable structural transformation and the formation of cubosomes to be determined in real time during the digestion of the optimal lipid composition. Imaging of the liquid-crystalline phases can also be achieved by vitrification of the samples at specific time points and then using cryotransmission electron microscopy (cryo-TEM) to image their morphology.23,24
■
EXPERIMENTAL SECTION
The dispersions were formed by dispersing the lipid mixture (phytantriol with triglycerides (purchased from TCI Chemicals, Tokyo, Japan) at required w/w ratios) in Pluronic F108 (BASF) solution in Milli-Q water (1% w/w) using ultrasonication (see ref 24 for additional details and suppliers of materials) such that all dispersions contained 90% w/w Pluronic solution/10% w/w total lipid. In vitro digestion experiments were conducted using a thermostated glass digestion vessel at 37 °C, fitted with tubing to enable flowthrough scattering analysis to be conducted in real time. Pancreatin (Southern Biological, Nunawading, Australia) at a concentration of 10 000 TBU 5374
dx.doi.org/10.1021/la5003447 | Langmuir 2014, 30, 5373−5377
Langmuir
■
Letter
RESULTS AND DISCUSSION The incorporation of TA into phytantriol cubosomes did not affect the nanostructure of the cubic phase even on substitution of 90% (w/w) of the phytantriol with TA (Figure 2). TA was
Figure 3. Time-resolved intensity vs scattering vector SAXS profiles during the digestion of a dispersion consisting of 15% (w/w) TB in phytantriol. The transformation from the disordered L2 phase to the highly ordered cubic phase at approximately 160 s is apparent from the development of Bragg peaks which index to the double diamond Pn3m bicontinuous structure. The process can be viewed as a video in the Supporting Information, and for clarity, the individual scattering profiles are shown in Supporting Information Figure S1 at t = 0 and 600 s.
Figure 2. Effect of incorporation of triglycerides (triacetin, TA; tripropanoin, TP; tributyrin, TB) into phytantriol cubosomes on the equilibrium internal order of the particles. Triglycerides were mixed with phytantriol prior to dispersion in 1% Pluronic F108 solution to provide steric stabilization. The arrow indicates the proposed trajectory through the phase diagram when a dispersion of 15% TB in phytantriol is subjected to lipolysis, leaving only phytantriol forming the particles.
situation where the hydrophobic triglyceride no longer occupies space in the bilayer, allowing a less-negative curvature to be attained. The experiments were conducted under a controlled pH stat condition at pH 6.5, which reflects the typical intestinal pH where lipolysis primarily occurs.29 The pKa of butyric acid is approximately 4.8,30 hence at pH 6.5 the liberated ionized fatty acid is expected to almost completely partition away from the lipid environment into the surrounding aqueous compartment, although some may remain, leading to the slightly lower lattice constant compared to that for the cubic phase formed by phytantriol alone in water. The changes in scattering are supported by the cryo-TEM images in Figure 4 that indicate a transformation from particles with a similar appearance to regular emulsion droplets prior to digestion (panel A) to faceted particles consistent with the cubic internal phase as would be expected after digestion is complete (panel B). Incredibly, virtually every particle in the field of view in the inset of panel B possessed some faceted morphology. The consistency of particle size in the imaging before and after digestion is also evident from the dynamic light scattering data in Table S1 in the Supporting Information, which shows that the mean particle size remained consistent at approximately 300 nm throughout the digestion process. Although some loss in total lipid volume within the particles is expected during the digestion process as the butyric acid and glycerol products move to the aqueous phase, this loss of material, which would lead to a decrease in particle volume and hence measured diameter, is expected to be countered by the influx of water on formation of the bicontinuous cubic phase, anticipated to contain approximately 30−35% water at equilibrium. A large population of coexisting, often adherent, vesicles is usually encountered when preparing cubosomes using topdown approaches. Extreme approaches such as placing dispersions in an autoclave after preparation have been proposed as a means of removing the “contaminating” vesicles.31,32 Interestingly and importantly, there were no vesicles apparent in the cryo-TEM images after digestion; the wide view in Figure 4 panel B best illustrates this point. The
presumed to be too hydrophilic to incorporate substantially into the lipid bilayer and so was not considered further. The addition of TP induced the formation of mixed cubic and inverse hexagonal phases between 5 and 15% (w/w) triglyceride and the H2 phase alone between 15 and 30% (w/ w) but required greater than 30% (w/w) to form a disordered inverse micellar structure. In contrast, TB had a strong influence on the phase behavior of the PHYT dispersions; the addition of just 5% (w/w) TB induced a transition to the inverse hexagonal phase, and at 15% (w/w) substitution, the ordered structures were lost and a disordered inverse micellar phase was evident. On the basis of the equilibrium studies, a dispersion containing 15% TB in PHYT was selected for the proof of concept demonstration of in situ cubosome formation on digestion of the triglyceride. The dispersion was subjected to the in vitro lipolysis model as described previously for studying the digestion of cubosomes prepared using the digestible lipid glyceryl monooleate20 and was similar to that used by others studying structure formation during triglyceride digestion.11 Remote addition of the enzyme allowed the fast kinetic resolution of changes in the scattering profiles; the TB-PHYT particles gave characteristic broad scattering from the disordered L2 phase, but at 160 s, there was a clear transformation to the highly ordered “double-diamond type” bicontinuous cubic phase (V2 Pn3m) (Figure 3), identifiable from the spacing ratios of the six peaks at 21/2, 31/2, 41/2, 61/2, 81/2, and 91/2. The L2 phase vanished by 190 s as indicated by the disappearance of the diffuse scattering at q ≈ 0.16 Å−1. Tributyrin is known to be digested quantitatively to three molecules of butyric acid and one molecule of glycerol under the action of lipase.28 As a result of the digestion of the hydrophobic triglyceride, the relatively hydrophilic digestion product (butyric acid) is apparently released from the lipid bilayer. In agreement with this scenario, the dimensions of the cubic lattice thereby increased from 60.7 Å at 160 s to 63.5 Å at 1020 s, trending toward that for dispersions of phytantriol alone (typically above 65 Å), which is to be expected in the 5375
dx.doi.org/10.1021/la5003447 | Langmuir 2014, 30, 5373−5377
Langmuir
Letter
Figure 4. CryoTEM images of dispersions comprising 15% TB in phytantriol before (panel A) and after (panel B) digestion. The inset of panel B illustrates the faceting of all particles in the dispersion and the complete absence of vesicular structures.
approaches in the assessment of in vivo pharmacokinetics of drug delivered from such materials,35 we will focus on the in vivo impact of these transformations in our next efforts.
novel enzymatic process presented here completely avoids their formation because there is no lamellar phase in the equilibrium phase behavior. Residual collagen strands from the enzyme are evident in the bottom left corner of Figure 4B; while residual enzyme may not be desirable depending on the application, there is no apparent limitation to the use of an immobilized enzyme in generating the structures using this approach, which could then be removed at a later time after the reaction is complete. Although in our experience being less efficient than using a pancreatin suspension, the immobilized enzymes are widely used in industry for the catalytic hydrolysis of triglycerides. We do not yet fully understand the mechanism by which the hydrolysis takes place when this enzyme is recognized as an interfacial enzyme; however, collision between droplets and immobilized enzyme is likely, and in the case of tributyrin as the substrate, some free tributyrin in aqueous solution may also be hydrolyzed. The equilibrium phase diagram would indicate that we should have encountered the inverse hexagonal phase during the digestion of the tributyrin component. However, the formation of the hexagonal phase is disfavored in these systems,33 and we have previously encountered the absence of the H2 phase during nonequilibrium processes including the cooling of cubosome dispersions and in the mixing of lipids between emulsions and cubosomes34 when the equilibrium behavior also suggested that it would be observed. The phenomenon is related to the geometric requirement for a void at the vertex of the hexagonal phase structure in a system with uniform amphiphile chain length and requires molecular distortion or space filling to occur, which is more readily achieved in an equilibrium system than in a dynamic system.33
■
ASSOCIATED CONTENT
S Supporting Information *
Dynamic light scattering description and data and a video illustrating the changes in internal structure occurring during lipolysis monitored using time-resolved small-angle X-ray scattering. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: +61 3 99039112. Fax: +61 3 99039583. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The studies were funded by the Australian Research Council through the Discovery Projects scheme (DP120104032). B.J.B. holds an ARC Future Fellowship. We are grateful to L. Waddington for assistance with cryo-TEM imaging. The SAXS experiments were undertaken at the SAXS/WAXS beamline at the Australian Synchrotron, Victoria, Australia.
■
REFERENCES
(1) Drummond, C. J.; Fong, C. Surfactant self-assembly objects as novel drug delivery vehicles. Curr. Opin. Colloid Interface Sci. 1999, 4, 449−456. (2) Angelova, A.; Angelov, B.; Mutafchieva, R.; Lesieur, S.; Couvreur, P. Self-Assembled Multicompartment Liquid Crystalline Lipid Carriers for Protein, Peptide, and Nucleic Acid Drug Delivery. Acc. Chem. Res. 2010, 44, 147−156. (3) Mezzenga, R.; Schurtenberger, P.; Burbidge, A.; Michel, M. Understanding foods as soft materials. Nat. Mater. 2005, 4, 729−740. (4) Nazaruk, E.; Bilewicz, R.; Lindblom, G.; Lindholm-Sethson, B. Cubic phases in biosensing systems. Anal. Bioanal. Chem. 2008, 391, 1569−1578. (5) Fraser, S. J.; Rose, R.; Hattarki, M. K.; Hartley, P. G.; Dolezal, O.; Dawson, R. M.; Separovic, F.; Polyzos, A. Preparation and biological
■
OUTLOOK The creation of highly uniform nanostructured particles was achieved by combining a lipid matrix with digestible components, providing a new approach to preparing dispersions with reduced energy input and potentially reduced requirements for the colloidal stabilizer. Perhaps more exciting is the evident potential for the design of such structure formation to occur in situ as a food component, with in vivo transformation to the nanostructured material and subsequent beneficial drug release behavior. Using our established 5376
dx.doi.org/10.1021/la5003447 | Langmuir 2014, 30, 5373−5377
Langmuir
Letter
evaluation of self-assembled cubic phases for the polyvalent inhibition of cholera toxin. Soft Matter 2011, 7, 6125−6134. (6) Kulkarni, C. V.; Ales, I. In Cubo Crystallization of Membrane Proteins. In Advances in Planar Lipid Bilayers and Liposomes; Academic Press: Boston, 2010; Vol. 12, pp 237−272. (7) Landau, E. M.; Rosenbusch, J. P. Lipidic cubic phases: A novel concept for the crystallization of membrane proteins. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14532−14535. (8) Shah, J. C.; Sadhale, Y.; Chilukuri, D. M. Cubic phase gels as drug delivery systems. Adv. Drug Delivery Rev. 2001, 47, 229−250. (9) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. r. Submicron Particles of Reversed Lipid Phases in Water Stabilized by a Nonionic Amphiphilic Polymer. Langmuir 1997, 13, 6964−6971. (10) Andersson, S.; Jacob, M.; Lidin, S.; Larsson, K. Structure of the cubosome - a closed lipid bilayer aggregate. Z. Kristallogr. 1995, 210, 315−318. (11) Salentinig, S.; Sagalowicz, L.; Leser, M. E.; Tedeschi, C.; Glatter, O. Transitions in the internal structure of lipid droplets during fat digestion. Soft Matter 2011, 7, 650−661. (12) Ljusberg-Wahern, H.; Nyberg, L.; Larsson, K. Dispersion of the cubic liquid crystalline phase - Structure preparation and functionality aspects. Chim. Oggi 1996, 14, 40−43. (13) Chemelli, A.; Maurer, M.; Geier, R.; Glatter, O. Optimized Loading and Sustained Release of Hydrophilic Proteins from Internally Nanostructured Particles. Langmuir 2012, 28, 16788−16797. (14) Nakano, M.; Teshigawara, T.; Sugita, A.; Leesajakul, W.; Taniguchi, A.; Kamo, T.; Matsuoka, H.; Handa, T. Dispersions of Liquid Crystalline Phases of the Monoolein/Oleic Acid/Pluronic F127 System. Langmuir 2002, 18, 9283−9288. (15) Spicer, P. T.; Hayden, K. L.; Lynch, M. L.; Ofori-Boateng, A.; Burns, J. L. Novel Process for Producing Cubic Liquid Crystalline Nanoparticles (Cubosomes). Langmuir 2001, 17, 5748−5756. (16) Abraham, T.; Hato, M.; Hirai, M. Glycolipid based cubic nanoparticles: preparation and structural aspects. Colloids Surf., B 2004, 35, 107−117. (17) Patton, J. S.; Carey, M. C. Inhibition of human pancreatic lipasecolipase activity by mixed bile salt-phospholipid micelles. Am. J. Physiol.: Gastrointest. Liver Physiol. 1981, 241, G328−G336. (18) Salentinig, S.; Phan, S.; Khan, J.; Hawley, A.; Boyd, B. J. Formation of Highly Organized Nanostructures during the Digestion of Milk. ACS Nano 2013, 7, 10904−10911. (19) Borne, J.; Nylander, T.; Ehan, A. Effect of lipase on different lipid liquid crystalline phases formed by oleic acid based acylglycerols in aqueous systems. Langmuir 2002, 18, 8972−8981. (20) Warren, D. B.; Anby, M. U.; Hawley, A.; Boyd, B. J. Real Time Evolution of Liquid Crystalline Nanostructure during the Digestion of Formulation Lipids Using Synchrotron Small-Angle X-ray Scattering. Langmuir 2011, 27, 9528−9534. (21) Angelov, B.; Angelova, A.; Filippov, S. K.; Narayanan, T.; Drechsler, M.; Štěpánek, P.; Couvreur, P.; Lesieur, S. DNA/Fusogenic Lipid Nanocarrier Assembly: Millisecond Structural Dynamics. J. Phys. Chem. Lett. 2013, 4, 1959−1964. (22) Angelova, A.; Angelov, B.; Garamus, V. M.; Couvreur, P.; Lesieur, S. Small-Angle X-ray Scattering Investigations of Biomolecular Confinement, Loading, and Release from Liquid-Crystalline Nanochannel Assemblies. J. Phys. Chem. Lett. 2012, 3, 445−457. (23) Spicer, P. T. Progress in liquid crystalline dispersions: Cubosomes. Curr. Opin. Colloid Interface Sci. 2005, 10, 274−279. (24) Mulet, X.; Kennedy, D. F.; Conn, C. E.; Hawley, A.; Drummond, C. J. High throughput preparation and characterisation of amphiphilic nanostructured nanoparticulate drug delivery vehicles. Int. J. Pharm. 2010, 395, 290−297. (25) Sek, L.; Porter, C. J. H.; Charman, W. N. Characterisation and quantification of medium chain and long chain triglycerides and their in vitro digestion products, by HPTLC coupled with in situ densitometric analysis. J. Pharm. Biomed. Anal. 2001, 25, 651−661. (26) Kirby, N. M.; Mudie, S. T.; Hawley, A. M.; Cookson, D. J.; Mertens, H. D. T.; Cowieson, N.; Samardzic-Boban, V. A low-
background-intensity focusing small-angle X-ray scattering undulator beamline. J. Appl. Crystallogr. 2013, 46, 1670−1680. (27) Hyde, S. T. Identification of lyotropic liquid crystalline mesophases. In Handbook of Applied Surface and Colloid Chemistry; Holmberg, K., Ed.; John Wiley & Sons: New York, 2002. (28) Wu, H.-S.; Tsai, M.-J. Kinetics of tributyrin hydrolysis by lipase. Enzyme Microb. Technol. 2004, 35, 488−493. (29) Kalantzi, L.; Goumas, K.; Kalioras, V.; Abrahamsson, B.; Dressman, J. B.; Reppas, C. Characterization of the Human Upper Gastrointestinal Contents Under Conditions Simulating Bioavailability/Bioequivalence Studies. Pharm. Res. 2006, 23, 165−76. (30) Riddick, J. A.; Bunger, W.B.; Sakano, T. K. Organic Solvents: Physical Properties and Methods of Purification, 4th ed.; John Wiley and Sons: New York, 1986; Vol. II. (31) Siekmann, B.; Bunjes, H.; Koch, M. M. H.; Westesen, K. Preparation and structural investigations of colloidal dispersions prepared from cubic monoglyceride-water phases. Int. J. Pharm. 2002, 244, 33−43. (32) Barauskas, J.; Johnsson, M.; Joabsson, F.; Tiberg, F. Cubic Phase Nanoparticles (Cubosome): Principles for Controlling Size, Structure, and Stability. Langmuir 2005, 21, 2569−2577. (33) Rappolt, M. The Biologically Relevant Lipid Mesophases as “Seen” by X-rays. In Advances in Planar Lipid Bilayers and Liposomes; Leitmannova-Liu, A., Ed.; Elsevier: Amsterdam, 2006; Vol. 5, pp 253− 283. (34) Tilley, A.; Dong, Y.-D.; Amenitsch, H.; Rappolt, M.; Boyd, B. J. Transfer of lipid and phase reorganisation in self-assembled liquid crystal nanostructured particles based on phytantriol. Phys. Chem. Chem. Phys. 2011, 13, 3026−3032. (35) Nguyen, T.-H.; Hanley, T.; Porter, C. J. H.; Boyd, B. J. Nanostructured liquid crystalline particles provide long duration sustained-release effect for a poorly water soluble drug after oral administration. J. Controlled Release 2011, 153, 180−186.
5377
dx.doi.org/10.1021/la5003447 | Langmuir 2014, 30, 5373−5377