256 aluminum inserts or inserts with a Hasteloy sintered ﬁlter (0.45 lm) pressed in the center and held in Bruker plastic sample cup holders were utilized for all the analyses. The water puriﬁcation system used was a Millipore milli-Q system. All other chemicals were obtained from Aldrich (St. Louis, MO) and were used without further puriﬁcation. A ten stage cascade impactor purchased from California Measurements (CA, USA) was used for aerosol particle size measurement. PARI LC jet nebulizer (Wuppertal, Germany) and BioAerosol Nebulizing Generator (BANG) from CH technology (NJ, USA) were used in our study. Jaeger-NYU Nose-Only Directed Flow Inhalation Expo- sure System from CH technology (NJ, USA) was used for aerosol animal dosing via a nose cone. Method Formulation A bench scale wet milling (micronization) device was used [23] with an appropriate amount of glass beads to produce a nanosuspension formulation of ﬂuticasone. Tween 80 at 0.5% (w/w) in phosphate buffered saline (pH 7.4) was added in a scintillation vial, and the mixture was stirred at 1,200 rpm for a period of 24 h with occasional shaking. The stock formulation was then harvested and potency was assessed by HPLC/DAD, and solid state checked by Powder X-ray diffraction (PXRD). Thermal gravimetric analysis with simultaneous differential thermal analysis (TGA/SDTA) was done on a Mettler TGA/SDTA851e. Particle size distribution was determined on a Beckman Coulter LS 230 particle size analyzer using the small vol- ume accessory (Miami, FL). A PIDS obscuration water optical model was employed. Particle size distribution was computed by the software using Mie scattering theory. The large particle size (D50 = 1.6 and D90 = 2.9 lm) ﬂuti- casone for in vivo comparison work was purchased from Sequoia Research Products. The formulation was made by directly suspending the bulk drug in 0.5% (w/w) Tween 80 in PBS. Potency, homogeneity, chemical stability, and solid-state stability were performed following the same procedure as listed above. Aerosol particle size measurement For the particle size distribution study, a ten-stage cascade impactor (GSI, California Measurement, Sierra Madre, CA., USA) was interposed between the nebulizer and the pump. The aerosol was generated using dry air at 50 psi. The impactor sampled the aerosol particles with aerody- namic diameters of 8.0 and 0.125 lm at a continuous ﬂow rate of 1 L/min-1. After a predetermined duration, the impactor was dismounted and different stages of the 123 Nanoscale Res Lett (2009) 4:254–261 cascade impactor were accurately weighted to determine the amount of deposition. The mass median aerodynamic diameter (MMAD) was calculated according to the percent cumulative accumulation at each stage. In Vivo and Sample Analysis The Pﬁzer Institutional Animal Care and Use Committee (IACUC) reviewed and approved the animal use in these studies. The Association for Assessment and Accreditation of Laboratory Animal Care, International fully accredits the Pﬁzer animal care and use program. Male Balb/c mice [8–10 weeks, *20 g] were used for all studies. For both the intratracheal (IT) and intranasal (IN) groups, the animals were anesthetized with an IP injection of a Ketamine/Xylazine mixture prior to the administration of 1 mg/Kg of Fluticasone in a nanosuspension and regular suspension. In the IT group, the anesthetized animals were held vertically with their mouths open. The tongue was pulled out gently to expose the trachea. Twenty-ﬁve microliters of the dose solution was pipetted into the trachea. For the IN group, the anesthetized animals were held vertically, and 25 lL of the dose solution was pipetted dropwise to the naris until it was completely inhaled. For the aerosol dosing arm, the conscious animals were exposed to the aerosol in a nose-only system for 10 min. The animals were sacriﬁced with an IP injection of an over- dose of Ketamine/Xylazine immediately after dosing. A blood sample was collected through cardiac puncture. Plasma samples were obtained by centrifugation at 8,000 rpm for 10 min, and 50 lL of the plasma sample was extracted with 100 lL of acetonitrile containing 0.05 lM of the internal standard (prepared in house). Samples were allowed to settle, and 50 lL was transferred to a 96-well plate. Analytical standards were prepared by spiking known amount of standards into control plasma and according to the above extraction procedure. Whole lung samples were collected, weighed, and kept on ice. In general, 200 mg of lung samples were ground in diluent containing 70% ACN and 30% water with the addition of 0.05 lM of internal standard (made in house) to a ﬁnal volume of 3.0 mL. Samples were allowed to settle, and 50 lL was transferred to a 96-well plate. Analytical standards were prepared by spiking known amounts of standards into blank lung tissue according to the above extraction procedure. The concentration of ﬂuticasone in all the samples was determined by LC/MS/MS on a Sciex API 4000 mass spectrometer in positive electrospray mode and MRM transition (m/z 502.1/313.7). Limit of detection (LOD) was 0.00015 lg/mL, and limit of quantiﬁcation (LOQ) was 0.0006 lg/mL. Compatibility of regular and nanosuspension of ﬂuticasone for aerosol delivery These experiments were conducted