Preparation, characterization and antibacterial activities of chitosan, N-trimethyl chitosan (TMC) and N-diethylmethyl chitosan (DEMC) nanoparticles loaded with insulin using both the ionotropic gelation and polyelectrolyte complexation methods

Abstract

TMC and DEMC, quaternized derivatives of chitosan, have been shown to have penetration enhancement properties and able to open the tight junctions of the intestinal epithelia at neutral and alkaline pH environments. The use of the nanoparticulate systems has the advantage of protecting the peptidic drugs from the harsh environment of the gastrointestinal tract. Hence, the aim of this study was to synthesize and characterize TMC and DEMC, both with quaternization degrees of 50 ± 5%, which were then used to prepare insulin nanoparticles with two different methods: ionotropic gelation and the polyelectrolyte complexation (PEC) techniques. The obtained nanoparticles were then characterized for size, zeta potential, insulin loading and release as well as antibacterial activities. The results showed that nanoparticles prepared by the PEC method had higher insulin loading efficiency and zeta potential than those made by the ionotropic gelation method and may subsequently be used for further in vitro, ex vivo and in vivo studies. Moreover, the antibacterial studies suggest that the polymers in free form have higher antibacterial activity against Gram-positive bacteria than in the nanoparticulate form.

Introduction

Oral drug delivery is the most favorite route for drug administration. In the past decade, biodegradable polymers such as chitosan and its quaternized derivatives have been studied extensively for their role as multifunctional permeation enhancers to enhance the permeation of hydrophilic macromolecules in peroral drug delivery. Chitosan, a natural polyaminosaccharide obtained from N-deacetylation of chitin, is a non-toxic, biocompatible and biodegradable polymer that has been used in biomedical fields in the form of sutures, wound covering, and as artificial skin (Dodane and Vilivalam, 1998, Domb and Bentolila, 1998, Varum et al., 1991, Rinaudo and Domard, 1989). Chitosan has mucoadhesive properties which are mediated by the spreading ability of chitosan over the mucus layer and additionally through its positive ionic interactions with the negative charges of the mucus or of the cell surface membranes (Lehr et al., 1992, Lehr et al., 1993). Furthermore, it has been shown that chitosan and its derivatives can act as antibacterial agents against both Gram-negative and Gram-positive bacteria (Henriksen et al., 1996, Jung et al., 1999). Chitosan is a polycation with an apparent pk_a 5.5; hence, in neutral and basic environments prevailing in the intestine, the chitosan molecules lose their charge and precipitation will occur. However, studies have convincingly shown that quaternized derivatives of chitosan, synthesized by introducing alkyl groups to the NH₂-group of the chitosan structure, are drastically more soluble in the neutral and alkaline environments of the intestine and hence more useful for drug delivery of peptides and proteins (Avadi et al., 2004, Thanou et al., 1999). Moreover, the soluble chitosan salts may act as permeation enhancers to increase the transmucosal absorption of peptide drugs that normally do not pass the tight junctional barrier. The enhancing properties of chitosan and its derivatives have been attributed to their interactions with the tight junctions and cellular membrane components to reversibly open the tight junctions and hence to increase the paracellular permeation of hyrodrophilic compounds (Park et al., 2002).

Today, biotechnological processes enable us to produce in large amounts endogenous peptides and proteins that are required for the treatment of chronic diseases. Although the peroral route for drug delivery is the most convenient and desirable one for patients, the harsh environment of the gastrointestinal tract, the high molecular weight of the peptides as well as their hydrophilicity which prevents them to cross the lipophilic barrier of the mucosal walls, are major problems in developing an effective delivery system.

Special strategies are hence required to overcome the above obstacles. It is the current opinion that nanosized polymeric particles, designed as delivery platform for those hydrophilic peptides, have the highest chance to succeed in overcoming these hurdles. Moreover, nanoparticles are capable of protecting drugs from degradation, improving permeation and penetration of the drugs across the mucosal surface as well as controlling the release of the encapsulated or adsorbed drugs (Schipper et al., 1997, Florence et al., 1995, Takeuki et al., 2001). The uptake of chitosan nanoparticles seems to be related to their size and the superficial charge; hence, the higher the superficial charge, the stronger the affinity of the nanoparticles for the negatively charged cell membrane (Janes et al., 2001, Sakuma et al., 1997). Various techniques are available for producing nanoparticles including solvent evaporation, interfacial polymerization and emulsion polymerization (Sakuma et al., 1999, Julienne et al., 1992, Ibrahim et al., 1992); however, most of these techniques involve the use of organic solvents, heat and vigorous agitation that may be harmful to peptides (Leroux et al., 1995). In 1997, the first chitosan nanoparticles were prepared by Alonso et al. using ionotropic gelation of chitosan (polycation) with tripolyphosphate TPP (polyanion). Ever since, almost all chitosan nanoparticles were prepared accordingly. However, recently polyelectrolyte complexation (PEC) resulting from self-assembly of proteins with natural and synthetic polymers has drawn increasing attention (Calvo et al., 1997). PEC is formed when oppositely charged polyelectrolytes are mixed and interact via electrostatic interactions. Both processes are easily applied; they have the advantage of not using sonication or organic solvents, both harmful for the proteins and peptides. The PEC formation results in an optically homogeneous and stable nano-dispersion (Mao et al., 2001). At a pH below 6.5, chitosan becomes positively charged due to protonation of the amino groups; on the other hand, most proteins become negatively charged at pH above 6.5. Hence, the electrostatic interactions between both entities at a suitable pH value can be used as driving force for PEC formation.

The aim of the present work was to develop nanoparticulate systems based on chitosan, diethylmethyl chitosan (DEMC) and trimethyl chitosan (TMC) using both the polyelectrolyte complexation and the ion gelation methods and to load the nanoparticles with insulin. The nanoparticles were characterized in terms of particle size, zeta potential, insulin loading and release as well as stability for further in vitro, ex vivo and in vivo investigations. Moreover, the antibacterial effects of these polymers were compared both in free polymer solution and in the nanoparticulate form.