Spray-drying of solutions containing chitosan together with polyuronans and characterisation of the microspheres

Abstract

Incubation of the rigid and transparent gel obtained upon pouring a chitosan hydrochloride solution into saturated ammonium hydrogen carbonate at 20 °C yielded chitosan carbamate, Chit-NHCO₂⁻NH₄⁺, soluble at alkaline pH values, typically 9.6. Addition of water to the gel isolated by centrifugation promoted the dissolution of the gel. By spray-drying the alkaline solution thus obtained, microspheres of chitosan were obtained. When chitosan carbamate was mixed with alginic acid, polygalacturonic acid, carboxymethyl cellulose, carboxymethyl guaran, acacia gum, 6-oxychitin, xanthan, hyaluronic acid, pectin, k-carrageenan, and guaran, clear solutions were obtained from which chitosan–polyuronan microspheres were easily manufactured by spray-drying. Those made of chitosan–xanthan or chitosan–guaran were unexpectedly found to be soluble in water; similarly, the chitosan–pectin microspheres were almost soluble. The microspheres containing hyaluronic acid or k-carrageenan underwent swelling when contacted with water; the other ones were insoluble. The microspheres were characterized by FTIR, X-ray diffraction spectrometry and scanning electron microscopy. The structural alterations detected were mainly due to interactions between the amino groups and the carboxyl groups.

Introduction

As a drug carrier, chitosan helps overcome certain adverse characteristics of drugs such as insolubility and hydrophobicity, but the semi-crystalline powder does not lend itself to direct compression. While chitosan powders have been evaluated in direct compression tests (Knapczyk, 1993), the formulations so far developed include excipients to facilitate compression (Rege, Shukla, & Block, 1999). A striking example are the commercial chitosan tablets for overweight control, which contain magnesium stearate as a binder, with negative consequences on the efficacy of chitosan.

Chitosan has been spray-dried not very often: chitosan suspensions (He et al., 1999, Davis, 1999, Rege et al., 2003a), chitosan salts (De la Torre, Enobakhare, Torrado, & Torrado, 2003), chitosan gelatin–ethylene oxide (Huang et al., 2002, Huang et al., 2003), and chitosan ethylcellulose mixture (Shi & Tan, 2002) have been assayed. Spray-drying of chitosan salts solutions provides chitosan microspheres having diameters close to 2–5 μm and improved binding functionality. The chitosan microsphere free-flowing powder is compressible and hence most suitable as a drug carrier (Rege et al., 2003a, Rege et al., 2003b, Sabnis et al., 1997).

The general chemical behaviour of chitosan, however, should be considered in order to avoid certain difficulties stemming from its insolubility at pH higher than 6.5 and its reactivity under the thermal conditions of the sprayer. For example, it seemed easy to spray-dry acetic solutions containing 1–2% chitosan at 168 °C, but the release of a drug from the spray-dried chitosan sharply depended on the acetic acid concentration because of the acetylation reaction occurring at that temperature. In fact, the degree of acetylation of chitosan increased during spray-drying and affected its enzymatic degradability (Shi & Tan, 2002).

Chitosan can be spray-dried at neutral pH if a colloidal suspension is prepared with NaOH. Nevertheless this preparation is time-consuming because it is difficult to wash the colloid and to remove excess alkali and salts.

As for alkaline media chitosan has been recently found to be soluble in NH₄HCO₃ solutions, where it assumes the ammonium carbamate form Chit-NHCO₂⁻NH₄⁺, i.e. a transient anionic form that keeps it soluble at pH 9.6, while reversibly masking the polycationic nature of chitosan. These original findings by Muzzarelli, Tosi, Francescangeli and Muzzarelli (2003) have as a valid background some works on glycoprotein synthesis (Likhosherstov et al., 1986, Linek et al., 1987, Manger et al., 1992, Cohen-Anisfeld and Lansbury, 1993, Merchan et al., 1997, Muzzarelli et al., 2002, Kunz et al., 1998, Vetter and Gallop, 1995, Tietgen et al., 2000, Wang et al., 2000, Ortiz Mellet et al., 1993, Lubineau et al., 1995). Because ammonium carbamates and NH₄HCO₃ decompose thermally and liberate CO₂, NH₃ and water, this alkaline system is perfectly suitable for producing chitosan microspheres by spray-drying.

It is known that chitosan forms polyelectrolyte complexes with polyanions, such as 6-oxychitin (Muzzarelli, Muzzarelli, Cosani, & Terbojevich, 1999), heparin (Kweon & Lim, 2003), carrageenan (Hugerth, Caram-Lelham, & Sundelof, 1997), pectin (Hoagland and Parris, 1996, Nordby et al., 2003), xanthan (Dumitriu, Magny, Montane, Vidal, & Chornet, 1994), acacia gum (Meshali & Gabr, 1993), hyaluronic acid (Laurent, 1998), alginic acid (Kim et al., 1999, Lai et al., 2003, Miyazaki et al., 1994), poly(acrylic acid) (De la Torre et al., 2003), carboxymethyl cellulose (Arguelles-Monal & Peniche-Covas, 1988), DNA (Rikimaru, Wakabayashi, Nomizu, & Nishi, 2003) and other macromolecules (Kubota and Kikuchi, 1998). These reactions are very fast and lead in general to the immediate disordered precipitation of the insoluble coacervates upon mixing, particularly when the final pH value is neutral or higher. On their part, the polyanions are normally in sodium salt form and show alkaline hydrolysis that contributes to chitosan insolubilization upon mixing.

As a consequence, chitosan–polyanion complexes have never been manufactured by spray-drying because chitosan itself is insoluble at pH values above 6.3; for instance, it is known that phase separation occurs when hyaluronan is mixed with chitosan particularly for stoichiometric ratios (SR=–NH₂/–COOH) close to 1.0.

The complex formation corresponds to charge neutralization at least partially, therefore the degree of complexation by charge neutralization is usually provided. For the system chitosan+hyaluronan the degree of complexation is ca. 1.0 regardless of the stoichiometric ratio value (provided SR>1); moreover, the degree of complexation is nearly independent of the degree of acetylation of chitosan up to 0.40. For higher degree of acetylation (randomly reacetylated chitosans) the complexation mechanism involves less cooperation. The chitosan–hyaluronan insoluble complexes are stable in acidic and alkaline media: they do not dissolve in NaOH, but dissolve in 0.2 M HCl. The complex is de-stabilized in NaCl brines (Rusu-Balaita, Desbrieres, & Rinaudo, 2003).

The scope of the present work is therefore to produce soluble polyelectrolyte complexes in alkaline solutions suitable for spray-drying, and then manufacture microspheres of chitosan–polyuronan complexes useful for subsequent drug delivery, thus overcoming the present limitations.

A number of anionic polysaccharides deserved immediate consideration, namely alginic acid, polygalacturonic acid, carboxymethyl cellulose, carboxymethyl guaran, acacia gum, 6-oxychitin, xanthan, hyaluronic acid, pectin, k-carrageenan, and guaran, that represent a selection of anionic polyelectrolytes capable of reacting with chitosan and currently studied in the food, pharmaceutical and medical fields (Collins, 1987).