Elsevier

Carbohydrate Polymers

Volume 92, Issue 1, 30 January 2013, Pages 33-39
Carbohydrate Polymers

Mucoadhesive nanoparticles made of thiolated quaternary chitosan crosslinked with hyaluronan

https://doi.org/10.1016/j.carbpol.2012.09.029Get rights and content

Abstract

Mucoadhesive polymeric nanoparticles intended for drug transport across the gastrointestinal mucosa were prepared from quaternary ammonium–chitosan conjugates synthesised from reduced-MW chitosan (32 kDa). Conjugates contained pendant moieties of 2–4 adjacent diethyl-dimethylene-ammonium groups substituted on repeating units (26–55%). Conjugates were thiolated via amide bonds with thioglycolic acid to yield products with thiol content in the 35–87 μmol/g range. Nanoparticles with mean size in the 270–370 nm range and positive zeta-potential (+3.7 to +12.5 mV) resulted from ionotropic gelation of the thiolated conjugates with de-polymerised hyaluronic acid (470 kDa). The nanoparticles were fairly stable in size and thiol content and showed a significant mucoadhesivity, matching and even exceeding that of the constituent polymers. Nanoparticles were internalised by endothelial progenitor cells in direct relation to their surface charge intensity. Nanoparticle uptake significantly improved cell viability and resistance to oxidation. The lyophilised nanoparticles were re-dispersible and could make a manageable formulation for oral use.

Highlights

► Thiolated quaternary chitosans were obtained from a chitosan of 32 kDa MW. ► Nanoparticles were obtained by interaction of these polymers with hyaluronan. ► The nanoparticles were modispersed and showed mucoadhesivity. ► The nanoparticles were internalized by endothelial progenitor cells. ► The nanoparticles improved cell resistance to oxidation.

Introduction

Mucoadhesive nanoparticulate systems for drug delivery by the oral route, that is the preferred route for systemic drug administration, have raised widespread interest for their recognised potential for improving the bioavailability of drugs with low mucosal permeability and/or lack of chemical stability in the gastrointestinal (GI) environment (Dünnhaupt et al., 2011, Ponchel et al., 1997, Sakuma et al., 2002, Sarmento et al., 2007, Takeuchi et al., 2001). Adhesion to mucus can slow down particle transit across the GI tract, thus prolonging residence of the carried drug at the absorption site and realising steep drug concentration gradients across the mucous membrane for a prolonged time (Dünnhaupt et al., 2011). Nanoparticles can also increase oral drug bioavailability by protecting the entrapped drug from degradation (Dünnhaupt et al., 2011, Ponchel et al., 1997, Sakuma et al., 2002, Sarmento et al., 2007). The prime consideration, when aiming at preparing mucoadhesive polymeric nanoparticles, is the basic material, which is supposed to be a mucoadhesive, biocompatible and biodegradable polymer. Chitosan derivatives positively charged on their repeating units are candidate materials in this respect. Our group have synthesised chitosan derivatives bearing short pendant chains containing a small number of adjacent quaternary ammonium groups, partially substituted on the chitosan repeating units (Zambito, Uccello-Barretta, Zaino, Balzano, & Di Colo, 2006). A comparatively high fraction of free, unsubstituted primary amino groups was left on the chitosan derivative backbone, available for covalent attachment of thiol-bearing compounds, via formation of 3-mercaptopropionamide moieties. This has, in fact, led to water-soluble thiolated quaternary ammonium–chitosan conjugates, the epithelial permeability-enhancing potential of which was tested using the Caco-2 cell monolayer and the excised rat jejunum as substrates (Zambito et al., 2009). On the basis of the obtained results the quaternary ammonium groups of these derivatives were ascribed the ability to reversibly open the epithelial tight junctions and also perturb the plasma membrane of the epithelial cells. On their part the thiol groups were believed to keep the polymer adherent to the epithelium by reacting with the thiols of the epithelial mucus to form disulphide bonds, thus favouring the permeability-enhancing action of the positive ions. This synergistic action points to the above chitosan derivatives as promising basic materials for preparing mucoadhesive bioactive nanoparticles. However, the polymers obtained by Zambito et al. (2009) have resulted, as such, unsuitable for this purpose, indeed, their ionotropic gelation with tripolyphosphate, used in the past to obtain nanoparticles from polycationic chitosan or N-trimethylchitosan (Sandri et al., 2007), yielded particles beyond the nanosize limit (unpublished results), possibly due to an exceedingly high molecular weight of the starting chitosan (590 kDa, viscometric). In fact it has been stated that 500 nm is the upper size limit for nanoparticles being able to pass across the intestinal mucus layer by endocytosis (Jani, Halbert, Langridge, & Florence, 1990).

Hence, the present work was aimed at (1) obtaining quaternary ammonium–chitosan conjugates starting from de-polymerised chitosan; (2) studying the effects of the reaction conditions, especially temperature, on the structures of products and their reproducibility; (3) introducing thiol groups on the above conjugates via formation of amide bonds with thioglycolic acid; (4) using the resulting thiolated derivatives to prepare stable nanoparticles of adequate size by ionotropic gelation with de-polymerised hyaluronic acid, which is a mucoadhesive polyanionic polysaccharide containing glucuronic acid units, and chemical crosslinking by formation of interchain disulphide bridges from thiol oxidation (Chang et al., 2012); (5) characterising nanoparticles for size and size stability, zeta potential, cytotoxicity, aptitude to be internalised by endothelial progenitor cells, mucoadhesivity compared to the component polymers; (6) planning a manageable formulation of nanoparticles for oral application.

Section snippets

Materials

The following materials were used. Thioglycolic acid (TGA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), fluorescein isothiocyanate (FITC), cellulose membrane tubing MW cut-off 12.5 kDa; (all from Sigma); 2-diethylaminoethyl chloride (DEAE-Cl) hydrochloride (Fluka); hyaluronic acid (HA), MW 950 kDa (Contipro, Dolní Dobrouč, Czech Republic); chitosan minimum 90% deacetylated from shrimp shell (Chitoclear FG90, Primex, Drammen, Norway). The commercial chitosan had an average

Synthesis of QA-rCh conjugates

The products obtained by reacting the reduced-MW chitosan (rCh) with DEAE-Cl had the basic structure of quaternary ammonium–chitosan conjugates, precisely, N,O-[N,N-diethylaminomethyl(diethyldimethylene ammonium)n]methyl chitosan, coded QA-rCh. They were similar in structure to the derivatives that were obtained by Zambito et al., 2006, Zambito et al., 2008 starting from commercial chitosan, having a much higher MW, as shown by the NMR spectra being similar (not reported). The conjugates would

Conclusions

The derivatives obtained by reacting de-polymerised chitosan with DEAE-Cl had the same N,O-[N,N-diethylaminomethyl(diethyldimethylene ammonium)n]methyl chitosan structure as those obtained starting from commercial chitosan, although the former had a much lower MW. The reaction temperature exerted a significant influence on such structural properties of the derivatives as the degree of substitution of the positively charged moieties on the chitosan repeating units and the number of adjacent

Acknowledgement

The work was funded by the University of Pisa.

References (22)

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