Investigation of lectin-modified insulin liposomes as carriers for oral administration

Abstract

The aim of this study was to design and characterize lectin-modified liposomes containing insulin and to evaluate the potential of these modified colloidal carriers for oral administration of peptide and protein drugs. Wheat germ agglutinin (WGA), tomato lectin (TL), or Ulex europaeus agglutinin 1 (UEA1) were conjugated by coupling their amino groups to carbodiimide-activated carboxylic groups of N-glutaryl-phosphatidylethanolamine (N-glut-PE). Insulin liposomes dispersions were prepared by the reverse-phase evaporation technique and modified with the lectin-N-glut-PE conjugates. Lectin-modified liposomes were characterized according to particles size, zeta potential and entrapment efficiency. The hypoglycemic effect indicated by pharmacological bioavailability of insulin liposomes modified with WGA, TL and UEA1 were 21.40, 16.71 and 8.38% in diabetic mice as comparison with abdominal cavity injection of insulin, respectively. After oral administration of the insulin liposomes modified with WGA, TL and UEA1 to rats, the relative pharmacological bioavailabilities were 8.47, 7.29 and 4.85%, the relative bioavailability were 9.12, 7.89 and 5.37% in comparison with subcutaneous injection of insulin, respectively. In the two cases, no remarkable hypoglycemic effects were observed with the conventional insulin liposomes. These results confirmed that lectin-modified liposomes promote the oral absorption of insulin due to the specific-site combination on GI cell membrane.

Introduction

Oral delivery is the preferred route of administration because it offers several advantages over other routes. It is more natural, less invasive, can be self-administered (outside the hospital), and is less expensive (Ahmed et al., 2002). However, oral delivery is generally not an effective method for the delivery of peptides and proteins.

The human gastrointestinal (GI) resists absorption of peptides, proteins and other large molecules until they are broken down into smaller molecules. The acidic environment of the stomach combined with an array of enzymes and physical barriers in the intestines either destroy or prevent efficient absorption of nearly all macromolecules. This problem leads to unacceptably low oral bioavailability (Lehr, 2000).

Several approaches to enhance the oral delivery of peptides have been or are currently being pursued. For instance, protective coatings, such as lipids and polymers, have been used to protect peptides during transport through the acidic environment of the stomach (Saffran et al., 1990) and enhance transport across the intestinal wall (Iwanaga et al., 1999). Alternatively, bioadhesive agents are used to enhance contact of the peptide to the intestinal wall (Ponchel and Irache, 1998, Arangoa et al., 2000). Such local delivery to sites allows greater adsorption and stability (Fara et al., 1985, Davis et al., 1988).

To achieve specific delivery of proteins and peptides across the intestinal epithelium after peroral administration receptor-mediated endocytosis can be utilized as a pathway. Recent investigations have focused on drugs conjugated with lectins.

There is evidence from the literature, that some plant lectins can facilitate the transport across cellular barriers (Clark et al., 2000). Lectins are proteins that recognize and bind to sugar complexes attached to proteins and lipids. They do this with very high specificity for the chemical structure of the glycan arrays.

The rationale behind lectin-mediated drug targeting is very simple (Bies et al., 2004). Most cell surface proteins and many lipids in cell membranes are glycosylated and these glycans are binding sites for lectins. The combination of a small number of sugars can produce a vast range of different chemical structures. Different cell types express different glycan arrays and in particular, diseased cells, such as transformed or cancerous cells, often express different glycans compared with their normal counterparts. Therefore, lectins could be used as carrier molecules to target drugs specifically to different cells and tissues. Apart from the concept of using the specificity of protein–sugar interactions for targeting to specific cells only, this kind of receptor-mediated bioadhesion may also be used to convey signals to cells in order to trigger vesicular transport processes into or across polarized epithelial cells. A number of studies have demonstrated the ability of lectins (agglutinins) to bind to intestinal mucosa.

Most cell surface proteins and many lipids in cell membranes of the GI are glycosylated and these glycans are binding sites for lectins. These glycans contain carbohydrate comprising N-acetyl-galactosamine, N-acetyl-glucosamine, galactose, fucose and sialic acid.

Wheat germ agglutinin (WGA) binds to N-acetyl-d-glucosamine and sialic acid exhibiting a molecular weight of 36 kDa. As compared with plant lectins with different carbohydrate specificity, the WGA-binding rate to intestinal cell lines of human origin, human colonocytes and prostate cancer cells was highest (Gabor et al., 1997, Gabor et al., 1998, Gabor et al., 2001). Moreover, the WGA not only binds to the cell membrane, but it is also taken up into the cytoplasm of enterocyte-like Caco-2 cells (Wirth et al., 1998).

Representing a dietary lectin, WGA is putative non-toxic. Wheat flour contains about 300 mg of WGA per kilogram and wheat germ is consumed either in unprocessed form as muesli or in processed form as bread. The muesli most likely contains the native lectin, bread is supposed to contain rather minimal amounts of intact lectin but the dietary intake of bread is rather high. Another hint towards negligible WGA toxicity is the observation that the viability of neither Caco-2 monolayers nor rats’ intestinal epithelial cells was reduced (Ishiguro et al., 1992). On the other hand, daily peroral administration of 42 mg WGA to rats for 10 days provoked antinutritive effects (Pusztai et al., 1993). It should be considered, however, according to a rough calculation this corresponds to a daily intake of 30 g pure WGA in man. To date, a final judgement on toxicity or atoxicity of WGA cannot be given due to lack of studies in vivo. Nevertheless, the amounts of lectins as necessary for glycotargeting of prodrugs or colloidal carrier systems are in the microgram range so that toxic effects should not be provoked (Gabor et al., 2004).

Tomato lectin (TL) had been shown to have specificity for N-acetylglucosamine and derivatives such as its tetramer (Kilpatrick, 1980). It was chosen because it was easy to purify, had been shown to bind to the intestinal mucosa of rats and was relatively resistant to degradation by intestinal enzymes. In addition TL was non-toxic to rats (Kilpatrick et al., 1985) and the fact that raw tomatoes were consumed by millions of people worldwide suggested it was not toxic to humans. Using radiolabelled TL, Naisbett and Woodley showed that it bound strongly to rat intestinal mucosa in vitro, targeting a number of the major glycoproteins of the intestinal brush border (Naisbett and Woodley, 1994a). As a consequence of binding to mucosal cells, it was also transported across the mucosa in vitro in significantly higher amounts than other macromolecules (Naisbett and Woodley, 1994b).

Ulex europaeus agglutinin 1 (UEA1), a lectin specific for a-l-fucose residues, binds almost exclusively to the apical surface of mouse Peyer's patch M cells in methanol or glutaraldehyde fixed tissues (Clark et al., 1993). Subsequent studies performed on freshly excised tissues or in ligated loops of anaesthetized animals revealed that lectin-binding to living follicle-associated epithelium (FAE) closely resembled that following tissue fixation (Clark et al., 1995, Giannasca et al., 1994, Gebert and Posselt, 1997). Moreover, UEA1 was successfully used to target macromolecules to mouse Peyer's patch M cells in gut loop experiments and to enhance subsequent macromolecule absorption across the intestinal epithelial barrier.

Using ovalbumin as a bystander, mistletoe lectin I strongly stimulated the immune response to the protein. In contrast, WGA and UEA1 exhibited slight adjuvant activity (Lavelle et al., 2001).

Thus the objective of this work is to get evidence whether WGA, TL or UEA1 can enhance the oral delivery of peptides. To follow this approach, model drug insulin had been incorporated in liposomes. Lectins were covalently bound to N-glut-phosphatidyl-ethanolamine (N-glut-PE) by carbodiimide techniques, then were incubated with insulin liposomes to obtain lectin-modified insulin liposomes. Lectin-modified liposomes were characterized according to particles size, zeta potential and entrapment efficiency. The stabilities were studied in pepsin solution and trypsin solution. The hypoglycemic effect of the lectin-modified insulin liposomes in diabetic mice was compared and the relative bioavailability in rats was evaluated.