Preparation and structural investigations of colloidal dispersions prepared from cubic monoglyceride–water phases

Abstract

Dispersions of bicontinuous cubic monoglyceride–water phases, so-called ‘cubosomes’, have been proposed as parenteral sustained release delivery systems. For the present study, dispersions of monoolein-rich monoglycerides (MO), with or without purified soya phospholipids (PL), were prepared by equilibration of a MO/(PL)/water cubic phase, subsequent fragmentation with a poloxamer 407 (P407) solution, sonication and homogenization. This yielded systems of very different macroscopic appearance: Almost transparent dispersions, slightly turbid systems, opaque dispersions or milky emulsions. The mean z-average particle diameters ranged from 80 nm to well above 350 nm. Considerable particle growth could be detected in most systems during storage at room temperature. Storage at 5 °C resulted in the formation of ointment-like gels, which may be attributed to the crystallization of MO. Freeze-fracture transmission electron micrographs of MO dispersions revealed predominantly spherical particles with a low fracturing tendency. Synchrotron radiation X-ray diffraction indicated that high energy input during disintegration of the cubic phase leads to very complex systems in which particles with a cubic structure and MO/(PL) vesicles may coexist. The characteristic reflections of cubic systems were absent in the diffraction patterns of almost transparent or slightly turbid dispersions. The results indicate a strong dependence of ultrastructure of the dispersions on the preparation parameters.

Introduction

Unsaturated long chain monoglycerides such as monoolein (MO) are able to form lyotropic liquid crystalline cubic phases in water. For monoolein–water-mixtures at room temperature there are two bicontinuous cubic phases in the concentration range between approximately 20 and 45% (w/w) water content corresponding to the space groups Ia3d (Q²³⁰) and Pn3m (Q²²⁴) (Qiu and Caffrey, 2000). Since the structures formed by the monoolein bilayers in these cubic mesophases correspond to specific infinite minimal periodic surfaces, IMPS, they are also often referred to as G-type (gyroid surface) and D-type (diamond surface; Hyde et al., 1984). For more complex systems, e.g. in the monoolein–poloxamer 407–water system, another mesomorphic cubic phase, Im3m (Q²²⁹) or P-type (primitive surface) has been described (Landh, 1994). Phospholipids (PL) can be incorporated into cubic mesophases (Landh, 1991).

The lipid bilayer units in simple MO/water systems form a three-dimensional network which separates two identical water-channel systems that have a water pore diameter of about 5 nm in the fully hydrated cubic phase (Landh, 1991).

Due to its lipid and water domains the cubic phase may in principle solubilize both water- and lipid-soluble substances, and molecules with amphiphilic character may partition at the lipid/water-interface. Biodegradability, the ability to incorporate and slowly release a variety of drugs with different physicochemical properties and the possibility to enhance the chemical, physical and/or enzymatic stability of incorporated drugs and proteins have made the cubic phase an interesting candidate for use in drug delivery (Engström, 1990a, Ericsson et al., 1991, Ganem-Quintanar et al., 2000, Shah et al., 2001, Wyatt and Dorschel, 1992). With respect to parenteral administration the use of cubic bulk phases is, however, limited by their high viscosity which makes them difficult to inject, and their general incompatibility with the intravenous route. Since bicontinuous cubic monoglyceride–water phases are stable in excess water and can be dispersed in appropriate surfactant and protein solutions (Lindström et al., 1981, Larsson, 1989, Ljusberg-Wahren et al., 1996), submicron-sized colloidal dispersions of these cubic structures, so-called ‘cubosomes’, have been proposed as parenteral sustained release delivery systems, especially for peptide and protein drugs (Landh, 1991, Engström, 1990b, Engström et al., 1996). It is assumed that the nanostructured lipid/water networks are preserved in the nanoparticles, and so are the properties of the cubic phase suggesting that the nanoparticles offer advantages similar to those of cubic bulk phases. Compared with liposomes, the high bilayer area to particle volume ratio of the cubic nanoparticles may increase the relative payload of lipophilic and amphiphilic drugs.

Several techniques have been described for the preparation of aqueous dispersions of cubic monoolein–water phases (Landh, 1991, Landh and Larsson, 1993, Gustafsson et al., 1996, Gustafsson et al., 1997, Nakano et al., 2001, Spicer et al., 2001). Information about the characteristics of monoolein dispersions is, however, still limited, particularly with respect to pharmaceutically relevant properties. The aim of the present study was to evaluate the production of cubosome dispersions following a procedure based on the original compositions and process of Landh and Larsson (Landh, 1991, Landh and Larsson, 1993), starting from an equilibrated monoolein–water bulk phase which is fragmented with poloxamer 407 and soya PL. Dispersion procedures with high energy input such as ultrasonication and high-pressure homogenization were applied to obtain particle sizes suitable for parenteral administration. The resulting dispersions were investigated with respect to storage stability, temperature sensitivity and physicochemical properties. The central questions were, first, whether the cubic structure is preserved during the dispersion process, and, secondly, whether it is able to exist in discrete particles of colloidal dimension for a sufficiently long time after dispersion of the original cubic bulk phase by high shear forces during homogenization. The structure of these colloidally dispersed monoolein–water phases was investigated by transmission electron microscopy (TEM) and synchrotron radiation X-ray diffraction. The use of synchrotron radiation permits systematic, in particular temperature dependent X-ray studies of the native cubosome dispersions.