In vitro characteristics of liposomes and double liposomes prepared using a novel glass beads method

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Abstract

A novel preparative method for liposomes and double liposomes (DL) using glass beads was superior to a glass-filter method developed previously. Lipid dissolved in chloroform was poured into a kjeldahl flask with glass beads (BZ-04, 0.350–0.500 mm φ; BZ-3, 2.794–3.962 mm φ; or BZ-6, 5.613–6.680 mm φ), and the organic solvent was evaporated. The lipid layer that formed on the glass beads was hydrated with 1.5 ml of the suspension of inner liposomes at a temperature above the phase transition temperature of the lipids employed, and was agitated vigorously. Erythrosine (ER) was used as a model drug. The size of liposomes prepared by the glass beads method depended on the size of the glass beads. The size of the liposomes became smaller as glass beads with a smaller size were used. A high encapsulation efficiency was observed when glass bead blends consisting of two different sizes were used. Large sizes (BZ-3/BZ-6) had a tendency to show high encapsulation efficiency and size also played an important role in the formation of liposomes. DL formation inhibited the release of ER and DL formative efficiency was markedly improved by means of the glass beads method. These findings suggested that the glass beads method developed in this study conferred a high drug loading and a high DL formation on liposomes compared with ordinary methods.

Introduction

Liposomes have received much attention as potential drug carriers for the improvement of intestinal absorption of drugs when taken orally [1], [2], [3], [4], and for delivery of the drugs into pathological sites such as tumors and inflammatory sites via intravenous administration [5], [6] by encapsulating drugs. Several studies regarding liposomes in an oral dosage form [1], [2], [3], [4] have been conducted since Patel and Ryman first reported in 1976 that the concentration of blood glucose was lowered after oral administration of liposomes encapsulating insulin to diabetic rats [7]. Recently, insulin- or salmon calcitonin-loading double liposomes (DL) prepared by the glass-filter method showed better hypoglycemic or hypocalcemic effects compared with ordinary liposomes in our group [8], [9]. As illustrated in Fig. 1, DL consists of a several small liposomes encapsulated in a large liposome [10], i.e. multivesicular vesicles (MVV) [11], [12], [13], therefore DL should be discriminated from ordinary classification (MLV, LUV and SUV). DL was estimated to be effective as a drug carrier for oral delivery compared with ordinary liposomes because the outer liposomes were suggested to protect the inner liposomes against several enzymes. However, the preparative method used in a previous study was not economic regardless of the contribution to the absorbability due to low encapsulation efficiency and low DL formative efficiency [9]. Thus, the development of a preparative method for efficient drug loading in inner liposomes and efficient DL formation was desirable. This study dealt with a novel preparative method for DL using glass beads.

Section snippets

Materials

Hydrogenated soybean phosphatidylcholine (H-soyaPC, peroxide value=0.4 meq/kg) and Coatsome EL series® were supplied by NOF Co. Ltd. (Tokyo, Japan). The following commercially available lipids and chemicals were used: dimyristoyl-phosphatidylcholine (DMPC), dimyristoyl-phosphatidylglycerol (DMPG), cholesterol (Ch) and egg yolk lecithin (EggPC) from Sigma Chemical Company (St. Louis, USA); stearylamine (SA) and erythrosine (ER) from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Glass beads

Evaluation of glass beads method (inner liposomes)

The encapsulation efficiencies of ER in liposomes prepared using glass beads (BZ-04, BZ-3 or BZ-6) with different amounts of DMPC (13, 26, 39, 52 or 78 μmol) are shown in Fig. 3. With any type of glass bead, ER encapsulation was enhanced with the increase in DMPC added.

Fig. 4 shows the size distribution of liposomes prepared using BZ-3 at 52 μmol of EggPC, DMPC or H-soyaPC. The mean diameters of liposomes, which consisted of EggPC, DMPC or H-soyaPC were 2.33, 2.14 and 2.48 μm, respectively,

Discussion

The glass beads method, which has a lipid membrane formed on the glass bead, differs from the glass-filter method, which has a lipid membrane formed on the glass-filter [10]. As indicated in Fig. 3, the encapsulation efficiency of ER in liposomes prepared using BZ-3 was higher than the others, i.e. 50.56% (P<0.05), in the case that 52 μmol of DMPC was used. For 26 μmol of the lipid, BZ-04 afforded a high drug encapsulation. Although the mechanism is not clear, it proved that the size of the

Conclusions

The glass beads method developed in this study afforded a high drug loading and a high DL formation to liposomes without special apparatus. It also exhibited good reproducibility, suggesting that liposomes could be prepared more easily and not expensively in comparison with ordinary methods. This study clarified that various kinds of liposomes could be prepared in shoals by altering the size of the glass beads and the kinds of lipid.

Acknowledgements

This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors wish to thank Ms Shio Kuroda for her technical assistance.

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