Oral insulin delivery using nanoparticles based on microemulsions with different structure-types: Optimisation and in vivo evaluation

Abstract

The purpose of this study was to optimise entrapment of insulin in poly(alkylcyanoacrylate) nanoparticles prepared from microemulsions with different microstructure containing isopropyl myristate, caprylocaproyl macrogolglycerides, polyglyceryl oleate and insulin solution and to investigate the in vitro release and bioactivity of insulin in nanoparticles dispersed in the microemulsion templates. Entrapment efficiency and release of insulin were studied using a reverse-phase HPLC assay. Morphology of the nanoparticles was examined with scanning electron microscopy. Bioactivity of insulin was studied using a streptozotocin-diabetic rat model. Nanoparticles were spherical with 200–400 nm in size without significant difference between different microemulsion templates, types and amounts of monomer. Entrapment efficiency increased significantly with increasing monomer concentration but decreased with increasing aqueous fraction in the microemulsion template. Insulin loading however, showed an opposite trend. In vitro release profiles of insulin from the nanoparticles dispersed in the microemulsion templates were controlled by the monomer concentration only. In vivo, a consistent and significant hypoglycemic effect over controls was found for up to 36 h depending on the type of monomer. No significant serum insulin levels were detectable. This study showed that the strategy of delivering insulin orally, entrapped in nanoparticles and dispersed in a biocompatible microemulsion is promising and highlights the importance of optimisation studies in combination with in vivo experiments.

Introduction

Many different strategies have been pursued to find ways to effectively deliver proteins and peptides via the oral route (Mahato et al., 2003, Morishita and Peppas, 2006). The major challenges therein are overcoming the gastrointestinal barriers and protecting the protein structure in the gastrointestinal tract. It has become the general opinion that it is unlikely for this goal to be achieved using only a single strategy, for example using permeation enhancers or enzyme inhibitors, and that combination strategies have been found more likely to be successful (Carino and Mathiowitz, 1999).

Promising results in oral delivery of proteins and peptides have been achieved using microemulsions that have absorption enhancing properties because of their components, mainly due to the oil and surfactants/cosurfactants (Ritschel, 1991, Spernath and Aserin, 2006). On the other hand microemulsions offer only limited, if any, protection of the peptide or protein against chemical and enzymatic degradation in the gastrointestinal tract. In contrast, particulate delivery systems can protect these problem drugs against degradation (Lowe and Temple, 1994) and oral uptake can be enhanced by translocation of the particles through the intestinal membrane (Aboubakar et al., 2000). In particular, poly(alkylcyanoacrylate) (PACA) nanoparticles have also demonstrated potential for oral delivery of bioactives due to their excellent biodegradability and biocompatibility (Lowe and Temple, 1994, Vauthier et al., 2003). The characteristics inherent to microemulsions and PACA nanoparticles imply that these formulation approaches in combination might be promising vehicles for the oral delivery of proteins and peptides. Firstly, PACA nanoparticles can conveniently be prepared by interfacial polymerisation using biocompatible microemulsions without the need to separate the resulting nanoparticles from the microemulsion template (Watnasirichaikul et al., 2000). Secondly, using different structure-types of microemulsions offers the flexibility to choose the microemulsion template that provides optimal loading and release (Krauel et al., 2005). It is possible that a difference in entrapment efficiency and loading will result from using different microemulsion templates, particularly between the water-in-oil-droplet type where the drug molecules are confined within the droplet around which the polymerisation occurs (Watnasirichaikul et al., 2000, Krauel et al., 2005), and the oil-in-water-droplet type, where the drug is solubilised in the continuous pseudo-phase.

A combination approach of oral delivery of insulin as a model protein entrapped in nanoparticles and dispersed in microemulsion templates with different microstructures was therefore chosen in this study. We have previously shown that a microemulsion system consisting of isopropyl myristate, caprylocaproyl macrogolglycerides, polyglyceryl oleate and water or a human insulin solution forms microemulsions with different structure-types, offering increased formulation flexibility for the solubilisation of drugs. These microemulsions were found to serve as templates for the formation of PACA nanoparticles resulting in nanoparticles with similar properties but only moderate entrapment efficiencies (Graf et al., 2008b). Optimisation of entrapment of the drug delivery system is thus desirable as a high loading capacity will reduce the amount of delivery system to be administered for the required dose. Although this may not be essential when using biocompatible components, a high entrapment efficiency is still advantageous from a cost-effectiveness point of view.

Alkylcyanoacrylates with a longer alkyl chain have a slower monomer-to-polymer conversion rate, yet have been reported to form a more highly interdigitated polymer network (El-Samaligy et al., 1986), and an increasing monomer concentration has been shown to improve entrapment of drugs (Radwan, 1995, Krauel et al., 2005). Therefore, the type and concentration of monomer as well as the type of microemulsion template may influence the entrapment and release rate of insulin. Increasing amounts of two monomers, ethyl (2) and butyl (2) cyanoacrylate, were used to prepare nanoparticles by interfacial polymerisation from water-in-oil (w/o), bicontinuous and oil-in-water (o/w) microemulsion templates to optimise insulin entrapment. Following in vitro evaluation of entrapment and release, samples with the highest entrapment efficiency and controlled drug release were tested for the in vivo activity of insulin in PACA nanoparticles dispersed in biocompatible microemulsion templates in a diabetic rat model.