Article pubs.acs.org/Langmuir
Facile Dispersion and Control of Internal Structure in Lyotropic Liquid Crystalline Particles by Auxiliary Solvent Evaporation Isabelle Martiel,† Laurent Sagalowicz,‡ Stephan Handschin,†,§ and Raffaele Mezzenga*,† †
Food and Soft Materials Science, Institute of Food, Nutrition & Health, ETH Zurich, Schmelzbergstrasse 9, CH-8092 Zurich, Switzerland ‡ Nestlé Research Center, Vers-Chez-Les-Blanc, CH-1000 Lausanne 26, Switzerland § Scientific Center for Optical and Electron Microscopy (ScopeM), Auguste-Piccard-Hof 1, 8093 Zurich, Switzerland S Supporting Information *
ABSTRACT: Submicron sized, structured lyotropic liquid crystalline (LLC) particles, so-called hexosomes and cubosomes, are generally obtained by high energy input dispersion methods, notably ultrasonication and high-pressure emulsification. We present a method to obtain dispersions of such LLC particles with a significantly reduced energy input, by evaporation of an auxiliary volatile solvent immiscible with water, e.g. cyclohexane or limonene. The inner structure of the particles can be precisely controlled by the addition of a nonvolatile oil, such as α-tocopherol or tetradecane consistently with bulk phase diagrams,. Two different lyotropic surfactants were employed, industrial grade monolinoleine (MLO) and soy bean phosphatidylcholine (PC). The lyotropic surfactant and oil phase modifier were first dissolved in the volatile solvent to give a liquid reverse micellar (L2) phase, which requires significantly less energy input to be dispersed in an aqueous solution of secondary emulsifier compared to the corresponding gel-like bulk mesophase. The auxiliary volatile solvent was then removed from the emulsion by evaporation at room temperature, yielding LLC particles of the desired inner structure, Pn3̅m, H2, or Fd3̅m. The obtained particles were characterized by small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). Our method enables fine-tuning of the final particle size through the volatile-to-nonvolatile volume ratio and processing conditions.
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INTRODUCTION Lyotropic liquid crystalline (LLC) particles are dispersions of bulk LLC mesophase in a continuous aqueous phase, stabilized by a secondary emulsifier. They offer promising prospects for delivery system applications, especially for drugs and nutrients in pharmaceutical and food systems.1,2 Thanks to the dual hydrophilic and hydrophobic character of LLC mesophases, and their large amount of interface, a wide variety of hydrophilic, hydrophobic, or even amphiphilic active molecules can be accommodated in the inner structure of LLC delivery systems.3 The promising potential of LLC mesophases encompasses biodisponibility4,5 and solubilization of active molecules such as phytosterols.2 Moreover, recent work indicates that stimuli-triggered phase transitions may help to achieve targeted controlled release of the active load;6−9 that is why structure control in LLC dispersions is of deep interest. A large variety of structures is found in both monoglyceridebased9−15 and PC-based16−23 systems upon addition of selected apolar components. To reduce the amount of surfactant used and the viscosity of the final applied formulations, dispersions of LLC particles are often preferred over bulk phases. Conventional dispersion methods for LLC particles such as direct ultrasonication and high-pressure emulsification imply high energy inputs.24−26 © XXXX American Chemical Society
Milder dispersion techniques are preferred in the presence of sensitive loads, such as proteins. The hydrotrope method proposed by Spicer et al.27 is a low energy dispersion method taking advantage of the miscibility of an auxiliary solvent (ethanol) in the continuous water phase. A large amount of ethanol is added to ease the dispersion by lowering the viscosity of the lipid mixture and bring about lipid droplet nucleation upon sudden addition of excess water.28 Variants of this method, involving other water-soluble auxiliary solvents such as propylene glycol and reduced amounts of ethanol, have been used to produce PC-based LLC dispersions by vigorous stirring, followed by a heat treatment to enhance particle monodispersity.18,29 The main drawbacks of the hydrotrope method are the difficulty to tune the particle size and the presence of ethanol in final samples, diluted in the water phase. Contact with ethanol and further heat treatments may denature proteins or damage sensitive loads. Furthermore, high energy input dispersion techniques are generally not adapted to PCbased mesophases, due to their high melting point and their tendency to form vesicles.16 Received: September 29, 2014 Revised: November 10, 2014
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dx.doi.org/10.1021/la5038662 | Langmuir XXXX, XXX, XXX−XXX
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surfactant (concentration comprised between 0.4 and 1.5 wt%), to reach typically 10% lipid phase, and ultrasonicated for 1 min with a UP200S sonicator (200 W, 24 kHz, Hielscher, Germany) set at 30% power, duty cycle 0.5. About 15 mL of the emulsion was transferred in a clean glass vial and finally left opened to evaporate at room temperature under the hood, under magnetic stirring. Total evaporation of the auxiliary solvent occurred typically overnight for cyclohexane, and within 2 days for limonene. Controlled Flow Conditions. To create a controlled atmosphere above the sample, in addition to the above description, the open vial was covered with a cap pierced with two holes. Compressed air with a constant pressure was bubbled through a thermostated Dreschel bottle containing water to fix the humidity level and the temperature.30 The bottle outlet was connected to one of the holes in the cap, while the second hole served as an outlet. As no precise measurement of the air pressure was available, the flow was quantitatively calibrated by measuring the evaporation of water from a blank F127 emulsion. For weighting the vial, the air inlet was briefly disconnected and the vial was weighted with its cap. The vial headspace height was about 2 cm. Small Angle X-ray Scattering (SAXS). Laboratory SAXS measurements were performed with a MicroMax-002+ microfocused X-ray machine (Rigaku), operating at 4 kW, 45 kV, and 88 mA. The Kα X-ray radiation of wavelength λ = 1.5418 Å emitted at the Cu anode is collimated through three pinholes of respective sizes 0.4, 0.3, and 0.8 mm. The scattered intensity was collected on a twodimensional Triton-200 X-ray detector (20 cm diameter, 200 μm resolution) normally for at least 30 min for bulk mesophases, respectively 2 h for dispersions. The scattering wave vector is defined as q = 4π sin(θ)/λ, where 2θ is the scattering angle. The SAXS machine is equipped with two sample chambers with different sampleto-detector distances, giving access to q ranges of 0.005−0.22 and 0.01−0.44 Å−1, respectively. Silver behenate was used for q vector calibration. Scattered intensity data were azimuthally averaged using SAXSgui software (Rigaku). Solid samples were loaded in a Linkam hot stage between two thin mica sheets and a rubber O-ring 1 mm spacer. Liquid samples were filled into 1.5 mm diameter quartz capillaries, sealed with epoxy glue (UHU). The X-ray machine is thermostated at 20 ± 0.1 °C, taken as room temperature. Particle Sizing. Dispersions were diluted 600-fold, and the intensity size distribution was measured by dynamic light scattering (DLS) using a Zetasizer Nano instrument (Malvern, U.K.) in backscattering mode (scattering angle of 173°). Each sample was measured three times (n = 3). The Z-average and polydispersity index (PDI) are intensity-based values derived from the cumulants analysis of the correlation data. Size distributions were essentially monomodal. When appropriate, intensity size distributions were converted in number size distributions using the refractive index of the droplets calculated from the tabulated refractive indexes of the components.37 Cryogenic Transmission Electron Microscopy (cryo-TEM). A FEI CM12 microscope was used for the cryo-TEM imaging. 300-mesh lacey carbon-coated copper grids (Quantifoil) were glow discharged (Emitech K100X, GB) for 45 s. A 2.5 μL portion of sample solution was applied onto the grids, and the excess of the dispersion was removed by a blotting paper. Liquid ethane cooled to −175 °C was used for sample vitrification. The vitrified sample was cryo-transferred into the cryo-TEM and kept at −180 °C during observation. Micrographs were recorded under low dose conditions (
