Pharmaceutical Nanotechnology
Starch-coated magnetic liposomes as an inhalable carrier for accumulation of fasudil in the pulmonary vasculature

https://doi.org/10.1016/j.ijpharm.2014.01.007Get rights and content

Abstract

In this study, we tested the feasibility of magnetic liposomes as a carrier for pulmonary preferential accumulation of fasudil, an investigational drug for the treatment of pulmonary arterial hypertension (PAH). To develop an optimal inhalable formulation, various magnetic liposomes were prepared and characterized for physicochemical properties, storage stability and in vitro release profiles. Select formulations were evaluated for uptake by pulmonary arterial smooth muscle cells (PASMCs) – target cells – using fluorescence microscopy and HPLC. The efficacy of the magnetic liposomes in reducing hyperplasia was tested in 5-HT-induced proliferated PASMCs. The drug absorption profiles upon intratracheal administration were monitored in healthy rats. Optimized spherical liposomes – with mean size of 170 nm, zeta potential of −35 mV and entrapment efficiency of 85% – exhibited an 80% cumulative drug release over 120 h. Fluorescence microscopic study revealed an enhanced uptake of liposomes by PASMCs under an applied magnetic field: the uptake was 3-fold greater compared with that observed in the absence of magnetic field. PASMC proliferation was reduced by 40% under the influence of the magnetic field. Optimized liposomes appeared to be safe when incubated with PASMCs and bronchial epithelial cells. Compared with plain fasudil, intratracheal magnetic liposomes containing fasudil extended the half-life and area under the curve by 27- and 14-fold, respectively. Magnetic-liposomes could be a viable delivery system for site-specific treatment of PAH.

Introduction

Over the past several years, magnetic nanoparticles have emerged as a new delivery tool for diagnosis, gene therapy, and targeted pharmacological interventions (Jain et al., 2008, Reiss and Hutten, 2005). In terms of size, these miniaturized particles closely resemble the mammalian cells (10–100 μm), viruses (20–450 nm), proteins (5–50 nm) and genes (2 nm wide and 10–100 nm long); and thus they can move around the body without interrupting physiological functions (Medeiros et al., 2011). Magnetic nanoparticles (MNPs) are chemically iron oxide (Fe3O4) and known as magnetites. They are biodegradable, biocompatible and possess super-paramagnetic properties (Okon et al., 1994). However, one of the major challenges toward using iron oxide based particles as drug delivery vehicles is their tendency to agglomerate and give rise to sediment. In fact, agglomeration of particles in the blood or at any major organ system could be a major safety concern (Medeiros et al., 2011). Coating of iron oxide particles with surfactants stabilizes against aggregation that limits the effect of gravitational sedimentation (Shamim et al., 2007, Shubayev et al., 2009). Similarly, coating with dextran confers stability and enhances the dispersibility of particulate systems in water (Bulte et al., 1999a, Jung and Jacobs, 1995).

To use magnetic particles as drug delivery carriers, drug candidates are often conjugated with coating materials such as dextran, starch, polyethylene glycol and block co-polymers (Alexiou et al., 2000). But coating of particles and conjugation of drug with coating materials can alter MNP surface and physical properties including magnetization, hydrodynamic size, charge and stability. Also, this approach can adversely influence biodistribution (Chouly et al., 1996), weaken drug–particle interaction and enhance drug–particle dissociation (Alexiou et al., 2000, Arias et al., 2008, Kim and Lee, 2001). Magnetic nanoparticles can also be encapsulated into various particulate carriers such as liposomes (Pradhan et al., 2010), polymeric nanoparticles (Li et al., 2011) and magnetic hydrogels (Mitsumata et al., 2012). Of the particulate carriers, liposomes are widely used microscopic spherical vesicles that can encapsulate both lipophilic and hydrophilic drug molecules in their lipid bilayer and aqueous core (Laouini et al., 2012). Magnetic particles encapsulated in liposomes maintain their magnetic property and exhibit improved pharmacokinetics profiles (Bulte et al., 1999b). They have been used for targeted delivery of drugs to the lung (Plank, 2008), kidney (Salomir et al., 2005), pancreas, brain (Kreuter, 2001, Schroeder et al., 1998) and colon (Duguet et al., 2006). Because of the presence of super-paramagnetic iron particles in the core, magnetic liposomes accumulate at the disease site and increase drug concentration under the influence of an external magnetic field (Shubayev et al., 2009). In hyperthermic condition, these particles produce fast drug release and increase drug concentration at the organ of interest (Plank, 2008, Shinkai et al., 2001) including the lung.

Indeed, the lungs are the primary site of a number of pathological conditions such as asthma, fibrosis, cancer, infection and pulmonary arterial hypertension (PAH). Of the lung diseases, PAH is a rare and progressive pulmonary vascular disease; proliferation and contraction of pulmonary arterial smooth muscle cells are chief pathological alterations that occur in PAH. Thus, it reduces pulmonary arterial lumen diameter, increases pulmonary vascular resistance, decreases reactivity of the vascular bed and eventually increases pulmonary arterial pressure (Mucke, 2008). Currently, endothelin receptor antagonists (e.g. ambrisentan and bosentan), prostacyclin analogs (e.g. epoprostenol, treprostinil and iloprost), phosphodiesterase-5 inhibitors (sildenafil), anticoagulants, calcium channel blockers, oxygen and nitric oxide (NO) are used for the treatment of PAH. However, these therapeutic agents are of short-lived and necessitate indwelling central catheters, dose escalation (Mucke, 2008), continuous dosing of longer acting prostacyclin analogs (Chattaraj, 2002) and frequent administration of nebulized formulations (Baker and Hockman, 2005). Furthermore, since current therapeutic agents lack pulmonary vascular selectivity (Nagaoka et al., 2005), peripheral vasodilatation and consequent systemic hypotension is common in PAH patients. Recent development in PAH therapy suggests that Rho kinase, a downstream effector of GTPase Rho, plays major roles in the pathogenesis of PAH. This enzyme is involved in smooth muscle contraction, cell adhesion, migration, cell growth and reorganization of actin cytoskeleton (Amano et al., 1997, Kimura et al., 1996). Thus, Rho-kinase inhibitors have come up as a new class of therapeutic agent for PAH. We and others have shown that fasudil, a Rho-kinase inhibitor, reduces pulmonary arterial pressure in PAH animals and human patients (Gupta et al., 2013, Oka et al., 2007). But lack of pulmonary selectivity remains to be a problem for this drug too.

To enhance pulmonary selectivity, we propose to develop a magnetic liposomal system that can be administered via the pulmonary route and that will help accumulate the drug in the pulmonary vasculature by means of an applied magnetic force. The proposed formulation is likely to release the drug slowly over a long period of time and produce sustained localized pulmonary arterial vasodilation. Toward this end, we have developed an MNP-based liposomal formulation of fasudil. We have encapsulated various iron oxide particles and fasudil in the liposomes and evaluated the feasibility of the resulting particles for inhalational delivery and assessed the influence of the magnetic field on the uptake of the particles by pulmonary arterial smooth muscles cells (PASMC). We have also studied particles’ influence on proliferation of PASMC. Finally, we have monitored the pharmacokinetics after intratracheal administration, and evaluated the safety with PASMC and Calu-3 bronchial epithelial cells.

Section snippets

Materials

Phospholipids were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma Aldrich Inc. (St Louis, MO). Anionic (Ferro Tec EMG 707) and cationic (Ferro Tec EMG 607) magnetic nanoparticles with an average particle size of 10 nm and saturation magnetization of 11 militesla (mT) were provided by Ferrotec Inc. (Bedford, NH) without any charge. Fluid Mag-HEAS, a commercially available ferrofluid containing Fe3O4

Physical characterization

Magnetic liposomes were round shaped (Fig. 1A) and magnetic particles were present both in the core and surface of the liposomes (Fig. 1B). The diameters of the liposomal particles were between 119 and 199 nm (Table 1). Entrapment of iron particles appeared to contribute to the size of the liposomes based on the observation that liposomes containing iron particles were larger than plain liposomes. Another important parameter, i.e. poly dispersity index (PDI) that is a dimensionless number that

Discussion

We and others have demonstrated the feasibility of liposomal formulation for intratracheal delivery of hydrophilic and hydrophobic small and/or macromolecules (Bai and Ahsan, 2010, Gupta and Ahsan, 2011, Gupta et al., 2013). It is now established that intratracheal administration of liposome can offer relative site-specificity and avoid off-target effects. However, a fraction of the intratracheally administered liposomes with a size range 100–200 nm may reach the blood circulation (Conhaim et

Conclusions

This study demonstrates the feasibility of magnetic liposomes as carriers for localized delivery of fasudil, an investigational anti-PAH drug, into the lungs. Indeed, favorable physicochemical properties, release kinetics, enhanced cellular uptake and anti-proliferative effect and extended half-life established the viability of magnetic liposomes for inhalational delivery. However, the efficacy of the formulations in reducing pulmonary arterial pressure and ameliorating pathogenesis in

Authors’ contributions

Ms. Nahar is the primary author of this article. She designed and performed all the experiments, analyzed data and prepared the draft of the article. Mr. Absar edited the article, wrote the introduction and discussion part. He also performed the microscopic study presented in this article. In addition, Shahriar helped the primary author in performing pharmacokinetic experiment and analyzing the PK parameters. Mr. Patel taught the primary author several analytical techniques and cell culture

Acknowledgements

We thank Drs. Hussaini Qhattal and Vinay Kumar of TTUHSC, Amarillo, TX, for their help in liposome preparation and fluorescent microscopic study. We also acknowledge Mr. Charles Linch at the Department of Medical Photography and Electron Microscopy, TTUHSC, Lubbock, TX, for his help with the transmission electron microscopy experiments. This work was supported in part by an American Recovery and Reinvestment Act Fund, 1R15HL103431.

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