M1a flowmeter

For examination of the aerodynamic properties, the multifunctional DPI formulations were applied to a Next Generation Impactor (NGI) (Copley Scientific, Nottingham, UK). Before the experiment, the impactor pans were coated with a Brij-coating consisting of 4 parts 15% Brij 35 in ethanol in 6 parts glycerol; 10 mL of Milli-Q water was filled into the pre-separator. For every experiment, a hard gelatin capsule (size 3) was filled with approximately 20 mg formulation. The air flow for application was set to 60 L/min, controlled by a M1A flowmeter (Copley Scientific, Nottingham, UK). The capsules were placed in a HandiHaler (Boehringer Ingelheim, Ingelheim, Germany) and were pierced. Aerosolization of the powder was achieved by applying a 4 second gas flow by a vacuum pump and critical flow controller (both Erweka, Heusenstamm, Germany). Then, the powders deposited in the different NGI cups were quantified by dissolving with a defined amount of water and analyzing the fluorescence signal of rhodamine B using a Tecan reader infinite 200 (Tecan, Männedorf, Switzerland). For each formulation, an individual calibration curve was prepared and the whole formulation was analyzed using an excitation wavelength of 565 nm and an emission wavelength of 625 nm. All experiments of every formulation were carried out in triplicate.