Nanoscale Res Lett (2009) 4:254–261 responsible for systemic side effects [6–9]. Due to the above ﬁndings, pulmonary targeting such as inhaled delivery is believed to provide an advantage over systemic administration, where the same degree of efﬁcacy can be achieved with lower systemic exposure. Aerosol pulmonary drug delivery is the preferred route of administration in the treatment of respiratory disease [10, 11]. Direct pulmonary delivery in humans is achieved using an aerosol generated by either an inhaler or nebu- lizer. The pharmaceutical industry is constantly seeking better and safer treatments for the pulmonary diseases. A major challenge in the identiﬁcation of pulmonary drug candidate is demonstrating pre-clinical efﬁcacy in appro- priate animal models with good translatability to humans. In order to predict accurately the efﬁcacy, delivery, dis- tribution, and PK of an inhaled drug, it is necessary to simulate the characteristics of the drug aerosol in pre- clinical models. Intranasal and intratracheal delivery of test compounds are often used to deliver drugs directly to the lungs of experimental animals. However, the above meth- ods are not practical in the clinical setting where an inhaler or nebulizer is used. Such a disconnect makes predicting PK/PD and efﬁcacious dose in humans very difﬁcult. A better pre-clinical modeling of drug delivery is necessary to obtain more accurate estimation of drug efﬁcacy. In this study, aerosol delivery in an animal model was investigated and ﬂuticasone propionate was used as a model compound. Fluticasone is a highly potent anti-inﬂammatory drug and is the most commonly prescribed inhaled glucocortioid. A nanosuspension formulation (D50 0.2 lm) was evaluated against regular suspension (D50 1.6 lm) for aerosol delivery. Nanosuspension has been widely used for drug delivery [12–16]. There are three key advantages of using nano- suspension formulations instead of solution formulations for pulmonary aerosol delivery. First, unlike solution for- mulations, nanosuspension formulations can be prepared without using large amount of organic co-solvents, thereby reducing the in vivo interference and potential toxicologi- cal effects caused by the co-solvents. Second, contrary to solution formations, the concentration of nanosuspension is not limited by solubility in the vehicle, thus, a wider dose range can be achieved. Finally, nanosuspension formula- tions provide superior content uniformity which results in greater conﬁdence of delivery and reproducibility. More- over, this mode of delivery can result in penetration of deep lung and smaller airways, leading to a more even drug distribution and resulting in a more accurate modeling of the drug distribution and efﬁcacy. In addition to the advantages conferred by nanosus- pension formulations, there are several reasons to choose aerosol delivery as well. Importantly, aerosol delivery has been widely used and evaluated for pulmonary drug 255 delivery in humans. We believe combining nanosuspension and aerosol in the pre-clinical setting will enable better and more consistent results can be generated. This advantage should allow us establish a more accurate modeling of the drug distribution and the resulting efﬁcacy. Despite the advantages of aerosol delivery, many factors can affect the outcome. Particle size, dosing system ﬂow rate, charac- teristics of the nebulizer, and drug solution concentration can often affect the characteristics of the aerosol and the delivery efﬁciency. In general, particles \5 lm are con- sidered suitable for inhaled drug delivery, and have a higher probability of being deposited in the airways of the lungs [17]. Despite these general understandings, conven- tional inhalers are found to be inefﬁcient and highly variable in delivering the desired dose to patients [18]. Many factors have contributed to the above ﬁndings. Devices such as pressurized and ultrasonic nebulizers produce widely dispersed particle sizes and operate con- tinuously, producing aerosol even when patients are not inhaling. This results in large transfer losses and poor lung deposition efﬁciencies [19] with the lung deposition being between 6 and 10% of the administered dose [20]. Despite the relatively low percent of deposition, the wide spread distribution of the particles in the lung results in equal or higher efﬁcacy as compared to intratracheal or intranasal methods [21, 22]. In this study, we pioneer the pre-clinical use of nanosuspension for aerosol delivery. The effects of various parameters are optimized to generate an aerosol capable of delivering a suitable dosage to support pre- clinical studies in rodents. Lung deposition, plasma exposure, impact on particle size, and delivery techniques are evaluated. Materials and Methods Materials Fluticasone propionate was purchased from Sequoia Research Products (Oxford, UK). HPLC grade acetonitrile was obtained from Burdick and Jackson (Muskegon, MI), and reagent grade formic acid, sodium hydroxide obtained from EM Science (Gibbstown. N J). The HPLC system used for formulation potency check was an Agilent HP 1100 HPLC equipped with a diode array (DAD) and a variable wavelength UV (VWD) detectors, and a quater- nary solvent delivery system (Palo Alto, CA). A Sciex API 4000 mass spectrometer (Applied Biosystems, Foster City, CA) coupled with HPLC was used for plasma drug analysis and quantiﬁcation. Powder X-ray diffraction (PXRD) was done on a Bruker D-8 Advance diffractometer for all the solid-state works to conﬁrm no form changes. A scintilla- tion counter was used for detection. In-house-fabricated 123