Pharmaceutical NanotechnologyPulmonary drug delivery with aerosolizable nanoparticles in an ex vivo lung model
Introduction
Pulmonary drug delivery offers several advantages in the treatment of respiratory diseases over other routes of administration. Inhalation therapy enables the direct application of a drug within the lungs. The local pulmonary deposition and delivery of the administered drug facilitates a targeted treatment of respiratory diseases, such as pulmonary arterial hypertension (PAH), without the need for high dose exposures by other routes of administration (Badesch et al., 2004). The intravenous application of short acting vasodilators has been the therapy of choice for patients with PAH over the past decade (McLaughlin et al., 2002). The relative severity of side effects led to the development of new prostacyclin analogues and alternative routes of administration (Rich and McLaughlin, 1999). One such analogue, iloprost (Ventavis®), is a worldwide approved therapeutic agent for treatment of PAH (Olschewski et al., 2002). Inhalation of this compound is an attractive concept minimizing the side effects by its pulmonary selectivity (Gessler et al., 2001). Unfortunately, the short half life of iloprost requires frequent inhalation manoeuvres, ranging up to 9 times a day (Gessler et al., 2008). Therefore, an aerosolizable controlled release formulation would improve a patient’s convenience and compliance.
Controlled drug delivery systems have become increasingly attractive options for inhalation therapies. A large number of carrier systems have been developed and investigated as potential controlled drug delivery formulations to the lung, including drug loaded lipid and polymer based particles (Zeng et al., 1995). Among the various drug delivery systems considered for pulmonary application, nanoparticles demonstrate several advantages for the treatment of respiratory diseases, like prolonged drug release, cell specific targeted drug delivery or modified biological distribution of drugs, both at the cellular and organ level (Pison et al., 2006, Sung et al., 2007, Azarmi et al., 2008, Yang et al., 2008). Nanoparticles composed of biodegradable polymers show assurance in fulfilling the stringent requirements placed on these delivery systems, such as ability to be transferred into an aerosol, stability against forces generated during aerosolization, biocompatibility, targeting of specific sites or cell populations in the lung, release of the drug in a predetermined manner, and degradation within an acceptable period of time (Rytting et al., 2008).
In order to combine the advantages of the direct access to the lung via inhalation with the unique properties of nanotechnology, the authors designed and characterized in vitro different nanocarrier systems for aerosol application, such as biodegradable nanoparticles (Dailey et al., 2003a, Dailey et al., 2003b). The most promising nanoparticle preparation has been formulated from a hydrophilic poly(lactide-co-glycolide) (PLGA) derivative, composed of short PLGA chains grafted onto an amine-substituted poly(vinyl alcohol) backbone, namely poly[vinyl 3-(diethylamino)propylcarbamate-co-vinyl acetate-co-vinyl alcohol]-graft-poly(d,l-lactide-co-glycolide), abbreviated as DEAPA. The amphiphilic properties of DEAPA make it highly suited for pulmonary formulations in several ways. On the one hand the flexibility of this type of polymer was shown through modifications in the degree of amine substitution and PLGA chain length, especially with regard to the controlled variability of biodegradation rates (Unger et al., 2008) and in vitro cytotoxicity (Unger et al., 2007). Moreover, this type of biodegradable polyester revealed no signs of inflammatory response in vivo (Dailey et al., 2006). On the other hand nanoparticles can be generated from DEAPA without the use of additional surfactant stabilizers. Furthermore, it has been reported that the addition of varying amounts of a polyanionic excipient, such as dextrane sulfate or carboxymethyl cellulose (CMC), to the polymer during nanoparticle formulation can generate nanoparticles of variable physicochemical properties. This attribute was used to design nanoparticle systems with greater stability in the face of shear forces and adjustable nanoparticle alveolar epithelial cell interactions (Dailey et al., 2003a).
Different methods are used to assess the behaviour of pulmonary administered drug loaded delivery systems ranging from in vitro to in vivo systems (Sakagami, 2006). Ex vivo isolated, perfused and ventilated lung models have been utilized in numerous pharmacological and toxicological studies to elucidate the fate of inhaled drug or toxic substances (Ewing et al., 2006, Ewing et al., 2008).
The purpose of the present study was to compare the pulmonary absorption and distribution characteristics of the hydrophilic model drug 5(6)-carboxyfluorescein (CF) after aerosolization as solution or entrapped into nanoparticles in an isolated, perfused and ventilated rabbit lung model (IPL). The preparation method of nanoparticles made up of a promising new class of biocompatible, fast degrading, branched polyester (DEAPA) loaded with CF by a modified solvent displacement technique is described. The particle size, particle size distribution, ζ-potential, particle morphology, drug encapsulation efficiency and in vitro drug release of the prepared CF loaded DEAPA nanoparticles were investigated using photon correlation spectroscopy (PCS), laser Doppler anemometry (LDA), atomic force microscopy (AFM) and fluorescence spectroscopy. Furthermore, the stability of the nanoparticles to the nebulization procedure utilizing an actively vibrating mesh device (Aeroneb® Professional) was determined using the above mentioned techniques. The aerosol characteristics of both formulations were analyzed by laser light scattering. Finally, the pulmonary dye absorption profiles for the two formulations after nebulization in an IPL were compared by monitoring the CF concentration in the perfusate over the time. Additionally, the dye distributions in the different compartments of the IPL at the end of the experiments were also evaluated.
Section snippets
Materials
The biocompatible, fast degrading, branched polyester, namely poly[vinyl 3-(diethylamino)propylcarbamate-co-vinyl acetate-co-vinyl alcohol]-graft-poly(d,l-lactide-co-glycolide), synthesized and characterized as described earlier by Wittmar et al. (2006) was used as polymeric nanoparticle matrix material. These polyesters were specifically designed for drug delivery to the lung and are comprised of short poly(d,l-lactide-co-glycolide) (PLGA) chains grafted onto an amine-substituted poly(vinyl
Nanoparticle characteristics
The measured physicochemical characteristics of the CF loaded DEAPA(39)-10 are summarized in Table 1. A representative AFM image of the freshly prepared nanoparticles is shown in Fig. 3a). Freshly prepared nanoparticles were of spherical shape with a mean particle size of 195.3 ± 7.1 nm (mean ± S.D., n = 4), a polydispersity index of 0.225 ± 0.017 (mean ± S.D., n = 4) and a ζ-potential of −28.3 ± 0.3 mV (mean ± S.D., n = 4), respectively. The preparation method assured an encapsulation efficiency of CF in the
Discussion
The CF loaded DEAPA(39)-10 nanoparticles were prepared by a modified solvent displacement technique. Due to their unique properties, such as biocompatibility and controlled variability of biodegradation rates, the DEAPA polymers are highly suited for pulmonary application (Dailey et al., 2005). Furthermore, their amphiphilic attributes facilitate nanoparticle generation without the requirement of surfactants. The addition of varying amounts of a polyanionic excipient, such as dextrane sulfate
Acknowledgements
The authors would like to thank Johannes Sitterberg (Department of Pharmaceutical Technology and Biopharmaceutics, University of Marburg) for his support with AFM and Nadine Faulstich (Medical Clinic II, Department of Internal Medicine, Justus-Liebig-University Giessen) for her technical assistance.
This study is part of the research project “Polymeric nano-carrier for pulmonary drug administration (Nanohale, FOR 627)” which is supported by the German Research Foundation (DFG). We want to
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