Ultrathin polyelectrolyte capsules for non-invasive delivery of proteins and peptides

Abstract

The development in genetic engineering has led to a proliferation of large quantities and varieties of proteins, which are highly pure and free from biological contaminants. In spite of their potency and specificity in physiologic functions, most of the protein therapeutics is difficult to administer clinically. Complexities of these agents demand an efficient carrier system so that the physiochemical and biological properties including molecular size, conformational stability, biological half-life, immunogenicity, dose requirements, complex feed back control mechanisms, susceptibility to break down in physical & biological environments, requirements for specialized transport mechanisms across biological membrane are duly controlled. In majority of the cases, chronic therapy of these peptides and proteins is warranted. Generally, they have an extremely short biological half-life. The inherent problems to parenteral protein delivery are as follows. 1) Patient compliance (repeated injections due to the short half-life). 2) Discomfort 3) Highly variable bioavailability (both within and between subjects for molecules such as subcutaneous insulin). 4) The non-physiological delivery pattern, particularly of subcutaneous injections. Further, little increase in actual drug delivered due to changes in dosage and/or mode of delivery may cause down regulation of the desired response to most of the proteins. In contrast, most often a pulsatile of flat delivery profile is required which mimics the normal physiological rhythm. To address these problems a great deal of research is being carried out to develop painless and convenient methods for delivery of proteins and peptides utilizing the approaches of novel drug delivery systems. During the past few decades, one of the major goals for many pharmaceutical scientists has been the discovery of a non-invasive means of administering protein/peptide(s). To achieve this goal attempts were made to deliver protein/peptides by various routes. 1.2 PER-ORAL DELIVERY OF PROTEINS AND PEPTIDES Whilst oral administration of insulin is a potentially attractive option, attempts to develop this route have, so far, met with little or no success [Berger, 1993]. Polypeptide drugs, such as insulin, are degraded in the acidic environment of the stomach and by digestive enzymes, especially in the small intestine. The epithelial surfaces of the gastrointestinal tract itself present an effective barrier to the absorption of insulin Table 1.1. Numerous individuals and combined strategies have been devised to enhance insulin absorption. These include the coadministration of insulin with enzyme inhibitors and/or permeation enhancers, methods to improve insulin’s chemical stability and the use of muco-bioadhesives, liposomes, emulsions and polymer-based delivery systems [Damgé, 1991]. Despite many different strategies tried, generally less than 1% of orally administered insulin is absorbed [Carino and Mathiowitz, 1999]. 1.2.1 Barriers for Oral Absorption of Protein/peptide(s) There are several extracellular and intracellular barriers to peptide/protein absorption. The oral bioavailability of most peptides and proteins is less than 1%. 1.2.1.1 Luminal proteases and intracellular peptides Peptide/protein hydrolysis is initiated in the stomach by pepsin I and II (Erickson and Kim,1990) and continues in the lumen of the small intestine by five potent pancreatic enzymes namely trypsin, chymotrypsin, elastase, carboxypeptidase A(CA-P) and B(CP-B). The specificities of these enzymes are complementary to each other and convert proteins into polypeptides, peptides and mixture of amino acids. 1.2.1.2 Gastro intestinal anatomical barriers The epithelial barrier rests on a basal lamina, a complex macromolecular structure that is likely to act as a barrier to proteins. (i) Oral and esophageal epithelium is made up of stratified squamous epithelium. In keratinized portion, the intracellular space constitutes oriented array of lamellae made up of neutral lipids with large proportion of ceramide. (ii) Gastrointestinal epithelium: It is lined with simple epithelium and consisted of columnar cells (enterocytes or absorptive cell). These cells are attached to each other by a complex apical junctional region (Terminal web). It consists of most apical zonula occludes (Tight junction) and distal zonula adherences (belt like dismosome attachment). They are attached to each other by inserted microfilaments of cytoskeleton. The apical membrane of enterocytes is thrown in to series of microvilli. The apical tight junction around the enterocytes plays a critical role by restricting paracellular pathway. Larger molecules may either diffuse across or be actively transported into enterocytes via lateral and basal membrane. The uptake is probably receptor-mediated endocytosis with ganglioside and galactoproteins of the membrane acting as specific ligands. (Linder et al., 1994). 1.2.2 Protein Absorption Mechanisms in the GI Tract Multiple transport mechanisms are involved in peptide and protein absorption in the GI tract. These mechanisms include (1) transcellular passage through lipid regions (2) transcellular carrier mediated transport (3) pinocytosis (or endocytosis) and (4) paracellular passage However, due to the large sizes of proteins, it is likely that they pass the epithelium paracellularly or through “pores”. Therefore, proteins from the extra cellular environment generally enter the cells via an endocytic mechanism although few may cross cell membranes directly (Doris & Maack, 1985). 1.2.2.1 Protein endocytosis Many proteins have been shown to enter the GI epithelium by endocytosis (Gonella & Newtra, 1984). There are essentially two types of endocytosis-Fluid phase endocytosis or pinocytosis (non-specific endocytosis) and adsorptive endocytosis (specific endocytosis). Nonspecific endocytosis is the engulfing of extracellular fluid containing dissolved proteins; specific endocytosis is a process of protein binding to the cell membrane followed by internalization of vesicles. Ligands can bind to either the apical or the basolateral membrane. These two membranes are separated by the tight junction. After internalization, membrane-bound ligands will be located in the apical or the basolateral endosome. Endosome-entrapped ligands can be further processed via the following pathways: (1) apical-to-basal or basal-toapical transcytotic pathway (2) endosome-to-lysosome pathways, followed by the degradation in lysosomes and releasing degraded products via exocytotic and (3) recycling pathways which may or may not involve the Golgi apparatus (Shen et al., 1992). Receptor mediated endocytosis is a special case of adsorptive endocytosis. Phagocytosis is engulfment of large particles (>0.5Hm) or molecular aggregates, that is also considered as one form of endocytosis (Steinman et al., 1983). However, this process is limited to specialized cells, such as macrophages and granulocytes. (a) Receptor Mediated Endocytosis The receptor-mediated process is common to virtually all eukaryotic cells except the mature erythrocytes (Stahl & Schwartz, 1986). The known receptors for mediating protein endocytosis are the GI tract includes those for EGF, immunoglobulin and transferrin. The process starts with the binding of macromolecules to cell surface receptors. These bound complexes then move and cluster within the coated pits of plasma membranes. The pits pinch off from the membrane and form coated vesicles for further intracellular processing. (b) Passive Diffusion Proteins may passively diffuse across the GI membranes and other tissues as well. For example, insulin appears to be transported at least partly by passive diffusion in rat intestine. There are two pathways of passive diffusion-Transcellular and Paracellular. The paracellular spaces are considered “pores” (Burton et al., 1991). The “pore” radius of the rat intestinal mucosal cells is 4Å for lipid insoluble non- electrolytes. (c) Para Cellular pathway Tight junctions have some epithelia that are sensitive to hormonal regulation and become leaky, facilitating paracellular pathway. This mechanism has gained interest because of low proteolytic activity. This depends on zonula occludens integrity and can be modified by lowering extracelluar Ca+2 or enhancing Na+ transport and/or glucose and amino acid transport. (d) Phagocytosis by intestinal macrophages Particles can be phagocytosed by gut macrophages within the intestinal wall and pass intracellularly to the mesentric lymph nodes (MLN). One Hm particles are transported (Wells et al., 1971) but quantitatively it is insignificant transport. (e) Protein Absorption in M Cells M cells, found in peyer’s patches are considered responsible for the most of the macromolecular absorption in the GI tract. This absorption is the initial stage for the initiation of host immune responses. In M cells, absorption of macromolecules by endocytosis is generally lower than in the rest of enterocytes. Protein loaded microparticles also absorbed largely in peyer’s patches than in non patches tissues. 1.2.2.2 Protein absorption via transcytosis Transcytosis refers to the transport of internalized vesicles carrying specifically or non-specifically adsorbed ligands or fluids from their sites of entry to sites on the opposite surface of the cell with subsequent release in a non-degraded form into the exracellular fluid. The main function of transcytosis is to redistribute macromolecules between different biological compartments and to maintain cell polarity.