Chitosan–PEG nanocapsules as new carriers for oral peptide delivery: Effect of chitosan pegylation degree

https://doi.org/10.1016/j.jconrel.2005.12.015Get rights and content

Abstract

We have previously reported the ability of chitosan nanocapsules to enhance and prolong the oral absorption of peptides. In the present work, our goal was to design a new type of nanocapsules, using chitosan chemically modified with poly(ethylene glycol) (PEG) (0.5% and 1% pegylation degree) and to investigate the consequences of this modification on the in vitro and in vivo behaviour of the nanocapsules. Chitosan–PEG nanocapsules and the control PEG-coated nanoemulsions were obtained by the solvent displacement technique. Their size was in the range of 160–250 nm. Their zeta potential was greatly affected by the nature of the coating, being positive for chitosan–PEG nanocapsules and negative in the case of PEG-coated nanoemulsions. The presence of PEG, whether alone or grafted to chitosan, improved the stability of the nanocapsules in the gastrointestinal fluids. Using the Caco-2 model cell line it was observed that the pegylation of chitosan reduced the cytotoxicity of the nanocapsules. In addition, these nanocapsules did not cause a significant change in the transepithelial resistance of the monolayer. Finally, the results of the in vivo studies showed the capacity of chitosan–PEG nanocapsules to enhance and prolong the intestinal absorption of salmon calcitonin. Additionally, they indicated that the pegylation degree affected the in vivo performance of the nanocapsules. Therefore, by modulating the pegylation degree of chitosan, it was possible to obtain nanocapsules with a good stability, a low cytotoxicity and with absorption enhancing properties.

Introduction

Despite the efforts dedicated over the last decades towards making the oral administration of peptides and proteins feasible, the actual fact is that this objective still remains a challenge. This is quite understandable if we take into account the great barriers that need to be overcome, such as the metabolic activity and the low permeability of the intestinal epithelium [1]. However, in spite of these difficulties, the success of the formulations undergoing clinical trials [2], [3], [4] offers an optimistic prospect towards reaching this objective.

The use of colloidal carriers such as nanoparticles and nanocapsules [5], [6], [7] has been reported to be a promising way to improve the oral bioavailability of peptides and proteins. The proposed mechanisms explaining the efficacy of these nanocarriers are: the great surface interaction of the nanocarrier with the absorptive epithelium, which can be further enforced by the use of bioadhesive materials, and the protective effect of the carrier for the associated peptide. Indeed, the effectiveness of nanocarriers at improving the absorption of labile macromolecules depends strongly on their polymer composition. For example, the simple surface modification of a nanocarrier using polymers with specific properties may be an easy approach to modulate its interaction with the epithelium. In this sense, an interesting polymer with a promising future in the biopharmaceutical and biomedical fields is the positively charged polysaccharide chitosan. In fact, chitosan has already been used in the form of nanoparticles and nanocapsules aimed at improving the transmucosal delivery of drugs across different mucosal surfaces [8], [9], [10], [11], [12]. Among these different transmucosal drug delivery applications, we have found the use of chitosan nanocapsules for oral peptide delivery quite promising [13], [14]. More specifically, following oral administration of chitosan nanocapsules containing salmon calcitonin, we have observed a great improvement on the pharmacological effect.

Taking this previous information into account, the main purpose of this work was to explore the possibilities to further improve the surface properties of chitosan nanocapsules in terms of their interaction with the gastrointestinal environment. For this purpose, we chose as an additional polymer, poly(ethylene glycol) (PEG). The choice of this polymer was justified because of two main reasons. First, the chemical modification of chitosan with PEG was seen as a way of improving the biocompatibility of chitosan [15]; second, we have previously observed that a PEG coating around the nanocarrier helps to improve their stability in the biological fluids and, as a consequence, facilitates the transport of bioactive macromolecules across the intestinal and nasal epithelia [10], [16], [17].

Therefore, the objectives of the present work were: first to obtain and characterize nanocapsules made of chitosan chemically modified with PEG, with two different substitution degrees (0.5% and 1%) and, second, to evaluate the effect of pegylation on the in vitro and in vivo behaviour of the resulting chitosan–PEG nanocapsules. More specifically, we evaluated the cytotoxicity of chitosan–PEG nanocapsules and their possible effect on the TEER of the Caco-2 cells monolayers and, finally, we investigated their efficacy at improving the intestinal absorption of the model peptide salmon calcitonin.

Section snippets

Materials

Salmon calcitonin was kindly donated by Almirall Prodesfarma, S.A. (Spain). Miglyol 812®, a triglyceride formed from medium chain fatty acids, was supplied by Lemmel (Spain). The surfactant soybean L-α-lecithin and Poloxamer 188 (Pluronic F-68®) were supplied from Sigma-Aldrich (Spain). Chitosan–PEG was synthesized from chitosan hydrochloride salt (Protasan® Cl110) which was purchased from FMC Biopolymer/Novamatrix, (Norway). Polyethylene glycol 5000 monomethyl ether (PEG) was supplied by Fluka

Results and discussion

We have recently shown that chitosan nanocapsules are able to enhance the nasal and the intestinal absorption of peptides [13], [14], [23]. On the other hand, previous work by our group has evidenced the positive role of a PEG coating around polyester nanocarriers with regard to their performance as transmucosal drug carriers [10], [16], [17]. More specifically, this PEG coating was found to enhance the stability of the nanocarriers upon contact with mucosal fluids and, their subsequent

Conclusions

In this paper we present chitosan–PEG nanocapsules, a new drug nanocarrier for the oral administration of peptides. The pegylation of the chitosan coating resulted in an improvement of the in vitro stability of the nanocarriers and also in an increase of the cellular viability of the Caco-2 cells. Moreover, these new nanocapsules resulted very efficient at increasing the intestinal absorption of the model peptide salmon calcitonin.

Acknowledgments

This work was supported by grants from the Spanish Ministry of Science and Technology (SAF 2000-0145 and SAF 2003-08765-C03-03) and Almirall Prodesfarma S.A. We thank María Isabel Loza for her advice for the cell culture experiments and Rafael Romero for his help with the animals' experiments.

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