258 The ﬁrst in vivo experiment was designed to compare different dosing techniques and impact of the nanosus- pension on lung deposition and systemic exposure. The target was focused on higher and dose depended lung disposition (to enhance local efﬁcacy) with lower systemic exposure (reduce systemic side effect). For this experi- ment, settings recommended by the manufacturer were used to test both PARI LC and BANG nebulizers. It was found that the PARI LC is more efﬁcient at a higher drug concentration with the lung deposition of 2.4 and 14.9 lg (approximately six folds) for the 1- and 5-mg/kg doses, respectively. The plasma exposures correspond to lung depositions with the evidence of reduced systemic expo- sures. For the PARI LC nebulizer, plasma concentration increase was observed when dose increased (for the 1- and 5-mg/kg doses: 0.01 and 0.02 lg/mL). However, the degree of increase is much smaller (only two folds) when compared with the lung exposure increase (approximately six folds). The dose-dependent increase in lung exposure along with lower systemic exposure was very much in accordance with the desired outcome. In comparison, the BANG devise lost efﬁciency at a higher dose with a deposition of 2.0 and 0.3 lg for the 1- and 5-mg/kg doses, respectively. The lack of delivery consistency at higher dose for BANG devise was reﬂected in plasma level as well. For the 1-mg/kg dose the plasma concentration was determined at 0.01 lg/mL and for the 5-mg/kg dose, plasma level was below the limit of detection. Based on the data, PARI LC nebulizer was chosen for further studies. The PARI LC devise, provided a dose-dependent drug increase in lung disposition and evidence of reduced sys- temic exposure (compare with the IT dose). Detailed information is illustrated in Figs. 2 and 3. Particle size distribution of the aerosol was further investigated by using a ten-stage cascade impactor with PARI LC nebulizer. The obtained data was used to further Nanoscale Res Lett (2009) 4:254–261 adjust the nebulizer to maximize the efﬁciency. Key parameters such as formulation concentration and system ﬂow rates were investigated and found to impact the aer- osol particle size and nebulizer efﬁciency. The system was optimized based on the best-obtained parameters. It was found when the optimized system was used, dose formu- lation, concentration, and ﬂow rate had minimal effect on the aerosol particle size. The MMAD was obtained by linearly ﬁtting the percent cumulative accumulation at each stage vs particle diameter. The obtained linear equations (y = ax ? b) were used to calculate the MMAD. In gen- eral, the MMAD of our optimized delivery setting was found to be about 3.7 ± 0.3 lm throughout a wide dose range (Table 1). The particle size of the nanosuspension formulation used for the studies is 0.2 lm (D50). A MMAD of 3.7 ± 0.3 lm indicates that aerosol particles are indeed aggregates and each aggregate contains from 17 to 20 nanoparticles. The obtained MMAD is well within the respirable range of an aerosol. Furthermore, in this study, the MMAD (\5 lm) of nanosuspension aerosol system is comparable to that of conventional aerosol sys- tem prepared from organic propellant [25, 26], which is clinically proven. Therefore, the delivery efﬁciency of nanosuspension is believed to be comparable to conven- tional systems. It is a viable option for pre-clinical drug delivery, and thus can provide a more suitable method for human dose projection. Several in vivo experiments were adopted. The ﬁrst experiment was designed to evaluate if regular suspension is suitable for in vivo aerosol delivery. The regular sus- pension was produced by using larger particle ﬂuticasone (particle size D50 to D90 1.6–2.9 lm). The particle size range used in this study is within the range used for dry powder inhaler. In general, when larger particles were used for aerosol delivery, the ﬂuticasone level was below LOD in both lung tissue and plasma. This ﬁnding was not a Effect of Dosing Technique on Lung Deposition Fig. 2 Effect of dosing technique on lung deposition 123 20 18 16 )g 14 µ ( en 12 o isca 10 t luF 8 f to 6 m A 4 2 0 1 mg/kg IN 1 mg/kg IT 1 mg/kg Aersol 5 mg/kg Aersol 1mg/kg Aersol 5 mg/kg Aersol BANG BANG PARI LC PARI LC