Pharmaceutical Nanotechnology
Systemic heparin delivery by the pulmonary route using chitosan and glycol chitosan nanoparticles

https://doi.org/10.1016/j.ijpharm.2013.02.035Get rights and content

Abstract

The aim of this study was to evaluate the performance of chitosan (CS) and glycol chitosan (GCS) nanoparticles containing the surfactant Lipoid S100 for the systemic delivery of low molecular weight heparin (LMWH) upon pulmonary administration. These nanoparticles were prepared in acidic and neutral conditions using the ionotropic gelation technique. The size and zeta potential of the NPs were affected by the pH and also the type of polysaccharide (CS or GCS). The size (between 156 and 385 nm) was smaller and the zeta potential (from +11 mV to +30 mV) higher for CS nanoparticles prepared in acidic conditions. The encapsulation efficiency of LMWH varied between 100% and 43% for the nanoparticles obtained in acidic and neutral conditions, respectively. X-ray photoelectron spectroscopy studies indicated that the surfactant Lipoid S100 was localized on the nanoparticle's surface irrespective of the formulation conditions. In vivo studies showed that systems prepared in acidic conditions did not increase coagulation times when administered to mice by the pulmonary route. In contrast, Lipoid S100-LMWH GCS NPs prepared in neutral conditions showed a pharmacological efficacy. Overall, these results illustrate some promising features of CS-based nanocarriers for pulmonary delivery of LMWH.

Introduction

Low molecular weight heparin (LMWH) is a linear anionic polysaccharide used as an anticoagulant for the prevention and treatment of deep vein thrombosis, pulmonary embolism and other thromboembolic disorders (Schulman, 2000, Yang et al., 2004). Unfortunately, LMWH exhibits poor oral bioavailability and, consequently, has to be administered via parenteral route. Due to poor patient compliance and side effects associated with injections, alternative routes for non-invasive LMWH administration have been actively investigated (Motlekar and Youan, 2006). Among those, the pulmonary route has attracted notable interest as a potential strategy to deliver therapeutically useful amounts of the anticoagulant (Qi et al., 2004). The large alveolar surface area available for drug absorption, the low thickness of the epithelial barrier, its extensive vascularization and relatively low proteolytic activity make pulmonary delivery of drugs of particular interest even for chronic therapy (Hussain et al., 2003, Labiris and Dolovich, 2003, Craparo et al., 2011, Licciardi et al., 2012). Moreover, it has also been observed that LMWH itself causes, upon pulmonary administration, a transient opening of the tight junctions in the lung epithelium, leading to a rapid onset of action and a Cmax comparable to subcutaneous administration (Qi et al., 2004). Nevertheless, beyond these positive aspects, it is believed that, without the use of penetration enhancers and adequate delivery vehicles, the amount of LMWH overcoming the pulmonary barriers and reaching the systemic circulation might be insufficient for an adequate pharmacological response.

Among the different pulmonary drug delivery vehicles, chitosan (CS)-based nanoparticles are particularly attractive. Indeed, besides promising properties including low toxicity, biocompatibility and high loading of hydrophilic molecules (Garcia-Fuentes and Alonso, 2012, Ieva et al., 2009), CS shows excellent mucoadhesive characteristics and it is capable of opening the tight junctions of epithelial cells, thereby improving the uptake of hydrophilic drugs (Mao et al., 2010). A CS derivative conjugated with ethylene glycol branches, i.e., glycol chitosan (GCS), which is water soluble at neutral and acidic pH values, has also been described (Siew et al., 2012). In addition to its adequate biocompatibility, GCS has been reported to retain the mucoadhesive properties inherent to CS (Trapani et al., 2009, Makhlof et al., 2010).

There are some previous reports on the potential of CS nanoparticles for the local and systemic delivery of macromolecules following pulmonary administration. For example, nanoparticles made of CS and hyaluronic acid (HA) have been used for local heparin delivery to the lungs (Oyarzun-Ampuero et al., 2009). The results of this work have shown that CS–HA nanoparticles are able to enhance the inherent ability of heparin to block the degranulation of mast cells (Oyarzun-Ampuero et al., 2009). Similarly, NPs made of CS or GCS have been recently described for pulmonary delivery of DNA (Bivas-Benita et al., 2004) and therapeutic peptides such as insulin (Al-Qadi et al., 2012) or calcitonin (Makhlof et al., 2010).

Taking this into account, the aim of the present work was to develop CS and GCS nanoparticles containing LMWH and to evaluate their performance following pulmonary administration. In addition, we found it critical to incorporate the non ionic surfactant Lipoid S100 to the nanoparticle's structure. Our hypothesis was that the co-encapsulation of LMWH and the penetration enhancer Lipoid S100 in CS and GCS nanoparticles would favour for the pulmonary absorption of macromolecules (Hussain et al., 2003). Moreover, Lipoid S100, being a mixture of natural phospholipids, was thought to improve the biocompatibility of the nanosystems in contact with the alveolar surface. Ultimately, we hypothesized that, by combining the mucoadhesive characteristics of the polymers with the nanoscale dimensions and the absorption enhancing properties of the surfactant and polymers, we could significantly enhance the pulmonary absorption of LMWH. In addition, nanoencapsulation was also conceived as a way to protect the anticoagulant drug from possible enzymatic degradation.

Section snippets

Materials

The following chemicals were used as received. Chitosan hydrochloride salt (Protasan, UP CL 113, Mw 110 kDa, deacetylation degree 86%, viscosity = 13 mPa/s according to manufacturer data sheet) was purchased from Pronova Biopolymer (Norway). Lipoid S100 was kindly donated by Lipoid KG (Germany). LMWH (average MW 18 kDa, 177 UI/mg), glycol chitosan (MW 400 kDa according to supplier instructions), mucine from porcine stomach (type II, bound sialic acid, ∼1%), glycerol, and pentasodium tripolyphospate

Preparation and characterization of LMWH-loaded CS and GCS nanoparticles

LMWH-loaded NPs were prepared by the well-known ionotropic gelation technique (Trapani et al., 2009). The two polysaccharides, CS and GCS, were dissolved in diluted NaCl (pH = 7) and acetic acid (pH = 4) aqueous solutions, respectively (Goycoolea et al., 2009, Trapani et al., 2009). The gelation of CS was induced by adding to the polycation solution a mixture of LMWH, Lipoid S100 and the polyanion TPP. The surfactant Lipoid S100 was dispersed in NaCl 87 mM prior to its addition to this mixture,

Discussion

In the context of anticoagulant therapy, the development of a non-invasive drug delivery system for LMWH could represent a useful approach to enhance patient compliance and minimize adverse effects. The purpose of this work was to evaluate the potential of CS and GCS NPs containing the pulmonary surfactant Lipoid S100 for the systemic delivery of heparin upon pulmonary administration. To this end, LMWH loaded CS- and GCS-based NPs were formulated using an adaptation of the ionic gelation

Conclusion

We have developed a novel nanocarrier consisting of Lipoid S100 and CS or GCS. These nanosystems, formed by ionic gelation technique, provided both sufficient entrapment efficiency and mucoadhesive properties. Aerosolization of these formulations indicated that heparin could be delivered to the lung. Overall, these nanocarriers might have a potential for systemic delivery of LMWH. However, further studies are required to elucidate the advantage of using this formulation for controlled release

Acknowledgments

We thank Prof. Maurizio Margaglione and the staff at the Medical Genetics Unit, University of Foggia (Italy) for their support with the coagulation tests and Prof. Pantaleo Bufo (Unit of Pathological Anatomy, University of Foggia, Italy) for histopathological analysis and imaging. Thanks are due to Lipoid KG (Germany) for a gift of Lipoid S100. We thank Dr. Sergey Lemeshko (NT-MDT Europe B.V.) for his help in the AFM analyses. MGF acknowledges a Parga Pondal Fellowship from Xunta de Galicia.

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These authors contributed equally to this work.

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