Chitosan–hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma

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Abstract

The purpose of this study was to produce mucoadhesive nanocarriers made from chitosan (CS) and hyaluronic acid (HA), and containing the macromolecular drug heparin, suitable for pulmonary delivery. For the first time, this drug was tested in ex vivo experiments performed in mast cells, in order to investigate the potential of the heparin-loaded nanocarriers in antiasthmatic therapy. CS and mixtures of HA with unfractionated or low-molecular-weight heparin (UFH and LMWH, respectively) were combined to form nanoparticles by the ionotropic gelation technique. The resulting nanoparticles loaded with UFH were between 162 and 217 nm in size, and those prepared with LMWH were 152 nm. The zeta potential of the nanoparticle formulations ranged from +28.1 to +34.6 mV, and in selected nanosystems both types of heparin were associated with a high degree of efficiency, which was approximately 70%. The nanosystems were stable in phosphate buffered saline (PBS), pH 7.4, for at least 24 h, and released 10.8% of UFH and 79.7% of LMWH within 12 h of incubation. Confocal microscopy experiments showed that fluorescent heparin-loaded CS–HA nanoparticles were effectively internalized by rat mast cells. Ex vivo experiments aimed at evaluating the capacity of heparin to prevent histamine release in rat mast cells indicated that the free or encapsulated drug exhibited the same dose–response behaviour.

Introduction

Although mast cells produce a variety of lipid mediators, chemokines, cytokines and enzymes that can interact with airway smooth muscle cells to cause hyperresponsiveness (Page et al., 2001, Robinson, 2004), they are the only endogenous source of heparin in mammals, which plays a protective role by limiting inflammation and airway remodelling (Page, 1991). Heparin is released on degranulation of mast cells (Green et al., 1993) and inhibits the proliferation of smooth muscle cells isolated from the airways of several species including humans (Johnson et al., 1995), bovines (Kilfeather et al., 1995) and dogs (Halayko et al., 1997).

Furthermore, several studies have demonstrated that the inhalation of high, medium and low-molecular-weight heparin (with or without anticoagulant activity) is effective in preventing acute bronchoconstrictor responses and airway hyperresponsiveness, with the potency of these types of heparin being inversely proportional to their molecular weight (Martinez-Salas et al., 1998, Molinari et al., 1998, Campo et al., 1999, Ahmed et al., 2000). This effect was attributed to the capacity of heparin to prevent mast cell degranulation. Interestingly, ultra-low-molecular-weight heparin was also effective in the treatment of late airway responses (pre- or post-antigen challenge); the effect was independent of the anticoagulant activity of the heparin and was mediated by an unknown biological action (Molinari et al., 1998, Ahmed et al., 2000). This is convincing evidence of the potential role of heparin in asthma therapy.

With the aim of enhancing the potential role of heparin in the treatment of asthma, we propose encapsulating this macromolecule in selected nanocarriers capable of positively interacting with mast cells, be internalized by these cells and released the encapsulated heparin in a controlled manner, thereby also preventing the possible degradation of the drug by enzymatic attack in the airways.

CS is a natural, non-toxic, biodegradable polycationic polysaccharide. We, and other groups, have previously used the polymer to elaborate different nanocarriers (Garcia-Fuentes et al., 2005, Köping-Höggård et al., 2005, Prego et al., 2005, De la Fuente et al., 2008a); these nanosystems have been shown, among other advantages, to prolong its residence time at the target site of absorption. These results were mainly attributed to the capacity of the polymer to interact with the negatively charged cell surfaces.

HA is a natural, non-toxic, biodegradable polysaccharide that is distributed widely throughout the human body, mainly in the connective tissue, eyes, intestine and lungs. Several ex vivo studies have demonstrated that particulate HA systems have beneficial effects on the mucociliary transport rate in airways, due to the mucoadhesivity of the polymer (Prichtard et al., 1996, Lim et al., 2000). It has also been found that HA has a discreet hypoproliferative effect on proliferating airway smooth muscle cells (Kanabar et al., 2005). This may also indicate that HA alone, or in synergy with heparin (Johnson et al., 1995), may be useful in preventing narrowing of the airway in asthmatic patients.

Taking into account this information and the previous experience of our group on the development of CS–HA nanoparticles loaded with hydrophilic and hydrophobic macromolecules (De la Fuente et al., 2008b), the present study aimed to combine the virtues of CS and HA in the development of heparin-loaded nanoparticles, intended for pulmonary administration. Finally, the interaction between these nanosystems and mast cells will be investigated, and their potential for preventing rat mast cell degranulation evaluated, to our knowledge, for the first time.

Section snippets

Materials

Ultrapure chitosan hydrochloride salt (CS; UP CL 113, molecular weight ∼125 kDa and degree of acetylation 14%) was purchased from Pronova Biopolymer AS (Oslo, Norway). Sodium hyaluronate ophthalmic grade (HA, molecular weight ∼165 kDa) was a gift from Bioiberica (Barcelona, Spain). Unfractionated heparin sodium salt (UFH, molecular weight ∼18 kDa, 202 USP units/mg), low-molecular-weight heparin sodium salt (LMWH, molecular weight ∼4 kDa, 53 USP units/mg) and pentasodium tripolyphosphate (TPP) were

Preparation and characterization of heparin-loaded CS–HA nanoparticles

Nanoparticles loaded with heparin were prepared by the ionotropic gelation technique. The ability of CS to form a gel after contact with polyanions by promoting inter and intramolecular linkages (Calvo et al., 1997) enables the formation of the nanoparticles. In this case, an ionic interaction occurs between the positively charged CS and the negatively charged HA, heparin and the polyanion TPP. The ionic gelation process is extremely simple and involves mixing two aqueous phases at room

Conclusions

Nanosystems were produced from CS and HA and their suitability as heparin carriers for the treatment of asthma was investigated. Confocal microscopy revealed that heparin-loaded CS–HA nanoparticles were internalized by rat mast cells. However, the capacity of free heparin and of heparin encapsulated in the nanosystems to prevent histamine release was very similar, and showed the same dose–response dependence.

Acknowledgements

The authors acknowledge financial support from the Spanish Government (SAF 2004-08319-C02-01 and Consolider-Ingenio CSD 2006-00012); Felipe Oyarzun-Ampuero was in receipt of a CONICYT scholarship. J.B. received financial support from the Programa Isabel Barreto (Xunta de Galicia). We also thank Mr. Salvador Arines for technical assistance with the mast cells assays.

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