Pulmonary toxicity and kinetic study of Cy5.5-conjugated superparamagnetic iron oxide nanoparticles by optical imaging

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Abstract

Recent advances in the development of nanotechnology and devices now make it possible to accurately deliver drugs or genes to the lung. Magnetic nanoparticles can be used as contrast agents, thermal therapy for cancer, and be made to concentrate to target sites through an external magnetic field. However, these advantages may also become problematic when taking into account safety and toxicological factors. This study demonstrated the pulmonary toxicity and kinetic profile of anti-biofouling polymer coated, Cy5.5-conjugated thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPION) by optical imaging. Negatively charged, 36 nm-sized, Cy5.5-conjugated TCL-SPION was prepared for optical imaging probe. Cy5.5-conjugated TCL-SPION was intratracheally instilled into the lung by a non-surgical method. Cy5.5-conjugated TCL-SPION slightly induced pulmonary inflammation. The instilled nanoparticles were distributed mainly in the lung and excreted in the urine via glomerular filtration. Urinary excretion was peaked at 3 h after instillation. No toxicity was found under the concentration of 1.8 mg/kg and the half-lives of nanoparticles in the lung and urine were estimated to be about 14.4 ± 0.54 h and 24.7 ± 1.02 h, respectively. Although further studies are required, our results showed that Cy5.5-conjugated TCL-SPION can be a good candidate for use in pulmonary delivery vehicles and diagnostic probes.

Introduction

For thousands of years humans have inhaled a variety of substances such as smoke, which achieves a systemic effect (Patton et al., 2004). Now, the medical establishment is beginning to realize the significant potential of this route for delivery of therapeutic agents. Pulmonary administration has become an alternative route of non-invasive systemic administration compared to the conventional gastrointestinal or intravenous routes (Edwards et al., 1997). Previous studies showed that pulmonary delivery of peptides and proteins exhibited higher rates of systemic absorption than other non-invasive routes (Jendle and Karlberg, 1996, Shen et al., 1999). Although there are still some concerns about pulmonary delivery such as the efficacy of delivery and the possibility of lung injury (Cleland et al., 2001), this route offers excellent advantages that include a larger surface area for absorption and extensive vasculature with thin epithelial barrier for easy permeation compared to conventional regimens (O'Hagan and Illum, 1990). Recent advances in the development of nanotechnology and devices now make it possible to accurately deliver drugs or genes to the lung. Nanoparticles as delivery vehicles present many advantages over other vehicles owing to longer retention time in the lung and decreased mucociliary clearance, allowing for the prolonged release of the loaded compound (Niven, 1995, Tsapis et al., 2002).

However, these advantages may also become problematic when regarding safety and toxicological issues. Previous studies showed that inhaled ultrafine particles could initiate a rapid onset of pulmonary inflammation, which can be associated with the development of lung fibrosis and tumors, in the long term (Dasenbrock et al., 1996, Driscoll et al., 1996). Furthermore, ultrafine particles have been shown to pass into systemic circulation and are linked to increased cardiovascular morbidity and mortality (Nemmar et al., 2001, Hamoir et al., 2003, Nemmar et al., 2003).

The choice of carrier used to deliver the drug to the lung is another important factor. Recently, the potential of magnetic nanoparticles as drug delivery vehicle was investigated (Lubbe et al., 1996, Alexiou et al., 2000, Jain et al., 2005, Alexiou et al., 2006, Kohler et al., 2006, Wang et al., 2007, Yu et al., 2008). The magnetic nanoparticles have many advantages including thermal therapy, guiding to the target site, and relatively non-toxic (Yu et al., 2008). Recently, Jon et al. reported anti-biofouling polymer coated, thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPION) as a novel diagnostic probe for in vivo cancer imaging (Lee et al., 2006, Lee et al., 2007).

The aim of this study was to evaluate the acute pulmonary toxicity and kinetic profile of Cy5.5-conjugated TCL-SPION by optical imaging. Furthermore, we elucidate the translocation pathway of the nanoparticles.

Section snippets

Synthesis and characterization of Cy5.5-conjugated TCL-SPION

Cy5.5 mono-NHS ester (2 mg) dissolved in 400 μL of dimethyl sulfoxide (DMSO) was added slowly to 1 mL of amino TCL-SPION (14 mg SPION/mL) (Lee et al., 2007) and allowed to react on ice in the dark with vigorous stirring. After 4 h, unreacted Cy5.5 was removed by gel filtration on Sephadex G-50. The resulting Cy5.5-conjugated TCL-SPION was stored at 4 °C in the dark until further use. Thermal gravimetric analysis (TGA) was carried out using TGA 2050 Thermogravimetric Analyzer (TA instruments).

Synthesis and characterization of Cy5.5-conjugated TCL-SPION

We prepared Cy5.5-conjugated TCL-SPION as described previously (Lee et al., 2006, Lee et al., 2007). The mean hydrodynamic size and its surface charge were measured by electrophoretic light scattering (ELS) and each value was 36 ± 8.1 nm and − 29.3 mV, respectively (Fig. 1A). The amount of polymer coating layer in the Cy5.5-conjugated TCL-SPION was determined with a thermal gravimetric analysis to be 20.2% (w/w) (Fig. 1B). Measurement of the number of dye molecules per milligram of TCL-SPION

Discussion

When Cy5.5-conjugated TCL-SPION enters into the lung, they are encounter with several different biological conditions including surfactant and lysosomal enzymes. In some extreme biological conditions such as several enzymes and acidic pH, the Cy5.5 dye might be released from the core nanoparticles. Therefore, confirming the stability of nanoparticles under the biological condition is very important. To confirm the stability of Cy5.5-conjugated TCL-SPION in biological condition, we monitored the

Acknowledgment

This work was supported by Nanotoxicology Program from the Korea Food and Drug Administration (09151KFDA693).

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