Stability of SUV liposomes in the presence of cholate salts and pancreatic lipases: effect of lipid composition
Introduction
In the last three decades liposomes have been extensively studied as possible delivery vehicles for drugs, enzymes, antibodies and genetic material (see Gregoriadis, 1995, Storm and Crommelin, 1998, for reviews). The majority of these studies involve the administration of liposomes intravenously or intraperitoneally. The potential for delivery of liposomal drugs by the oral route has not been well elucidated despite the fact that in the past there has been an increased interest in the use of liposomes as a highly convenient orally administered delivery system. Many agents such as insulin (Dapergolas and Gregoriadis, 1976, Patel and Ryman, 1981, Woodley, 1985, Choudhari et al., 1994, Takeuchi et al., 1996), and other proteins or vitamins (Arien et al., 1993, Tsume et al., 1996, Fukunaga et al., 1991, Maitani et al., 1996, Kirilenko and Gregoriadis, 1993) have been tested and though they were found to reach the blood circulation after oral administration of their liposomal preparations, their absorption results were mixed and unpredictable in most cases (especially in the earlier studies for insulin). However, the results of several studies involving oral administration of liposomal insulin indicate the occurrence of a marked hypoglycemic response. This has been linked, by some researchers, to the uptake of liposomes by enterocytes, which has been demonstrated in vitro by Rowland and Woodley (1981), while others claim that liposomes act as a barrier, protecting encapsulated molecules from degrading agents present in the gastrointestinal tract. Regardless of the controversy, a major prerequisite for effective oral administration of liposomal formulations, is their survival in the hostile environment of the gastrointestinal (GI) tract. In previous studies (Arien et al., 1993, Sehgal and Rogers, 1995, Ramaldes et al., 1996a, Ouadahi et al., 1998), the stability of liposomes in media mimicking the conditions of the GI tract, has been seen to increase when saturated phospholipids or high amounts of cholesterol were used for liposome preparation. Although most liposomal formulations previously studied have been found stable in media with different pH values and in the presence of pancreatic lipases, they were rapidly losing their entrapped material in the presence of cholate salts. In most of these studies the stability of liposomes was tested only for a short period of time (0.5 or 1 h), thought it is well known that the mean residence time of a formulation administered orally is well over 2 h. In addition, most of the previous studies involved multilamellar vesicles which are expected to be more stable (when stability is judged by retention of entrapped molecules) due to the many bilayers surrounding the encapsulated material. Also, these liposomes are larger in size; a factor that has been linked with particle uptake by enterocytes (Jani et al., 1990, Eldridge et al., 1990, Florence, 1997) as well as absorption of recombinant human erythropoietin after oral administration of liposomal formulations (Maitani et al., 1996). In the present study the membrane integrity of small unilamellar liposomes with different lipid compositions (measured as their ability to retain a small hydrophilic molecule, 5,6-carboxyfluorescein) was evaluated in the presence of cholic salts (sodium cholate and sodium taurocholate) and a mixture of pancreatic lipases, for a period of at least 5 h. The size distribution of several of the liposomes studied was measured before and after sodium cholate addition in the samples and also after a 24-h incubation period at 37°C, in order to further elucidate the mechanism of CF release from liposomes.
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Materials
Egg l-α-phosphatidylcholine (PC) (grade 1), dipalmitoyl-phosphatidylcholine (DPPC) (synthetic, grade 1), distearoyl-phosphatidylcholine (DSPC) (synthetic, grade 1), dimyristoyl-phosphatidylcholine (DMPC) (synthetic, grade 1), and sphingomyelin (SM) (from bovine spinal cord), were obtained from Lipid Products, Nutfield, UK. The 99% purity of the lipids was verified by thin layer chromatography as described by New (1990). All lipids used gave single spots on TLC plates. Cholesterol (Chol) (pure),
Liposome stability measured by CF release
In general the stability of liposomes in cholate and taurocholate salts is low, while most of the lipid compositions studied are stable in the presence of pancreatin. High amounts of the encapsulated CF (approaching in some cases 100%) were released from some of the liposomes studied, even after 1 h of incubation in the presence of cholates (both cholic and taurocholic salts).
More specifically, all liposomes containing DMPC are very unstable (data not shown) in the presence of cholates and
Discussion
The main objective of this study was to determine the possibility of formulating liposomes, which will (fully or partly) retain their integrity after oral administration. Therefore the membrane integrity of liposomes composed of several different lipids (with transition temperatures below and above 37°C) was followed after incubation in cholate or taurocholate salts, and also in a mixture of pancreatic lipases. Liposomes composed of plain lipids or mixtures of lipids with cholesterol, which is
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