In vitro particle deposition
The particle size on the basis of aerodynamic diameter and the deposition pattern of the prepared provesicular powder for pulmonary delivery are assessed using 8-stage non-viable Cascade Impactor at an air flow rate of 60 l/min for 4 s. All impactor plates are precoated with suitable solvents like silicone grease to minimize the particle bouncing after impaction. Prepared provesicular formulation was filled into hard gelatin capsule of size 2 and placed in the Handihaler, pierced and aerosolized for 4 s. Using the suitable rinsing solvent, the powder samples deposited in capsule and inhaler device, induction port, preseparator and on each plate of cascade impactor are washed and drug content is analyzed by HPLC. Total emitted dose gives the post-aerosolization weight difference between the initial weight of powder loaded into the capsule and the amount of powder retained within the handihaler. The mass median aerodynamic diameter (MMAD) was obtained from the log probability graphs. Fine particle fraction (FPF) was defined as the percent weight of aerosol powder particles with an aerodynamic diameter less than 5 m.
Optical microscopic evaluation is performed to understand the formation of vesicles (Liposomes/niosomes) from formulated provesicular (proliposomal/proniosomal) powders upon hydration (Janga et al., 2012). In this study provesicular powder is placed on a glass slide and hydrated by adding few micro liters of distilled water through the narrow slit between the slide and coverslip. The formation of vesicles under static conditions (without shaking and gentle shaking by manually with distilled water) is observed and photomicrographs are taken using optical microscope. The generation of vesicles is monitored for different provesicular formulations developed using different inert carriers with varying composition of lipid or surfactant/ cholesterol.
Transmission electron microscope (TEM)
The morphology and aggregation behavior of the vesicles formed by manual shaking of provesicular formulation with distilled water for a period of 2 minutes, are examined by performing transmission electron microscopy. A thin film of vesicular dispersion is made over a carbon-coated copper grid and allowed to stand for 5 minutes to adhere vesicles on the surface of the grid. The sample is stained negatively using sodium phosphotungstate solution (0.2% w/v) before observing and capturing of vesicle images under transmission electron microscopy.
Number of vesicles per cubic mm
With respect to the formation of vesicles from provesicular powder, number of vesicles formed is also one of the prime prerequisite in the optimization of the composition of the provesicular formulation. The vesicles obtained upon hydration of provesicular formulation with distilled water are mounted on a hemocytometer and counted by focusing under optical microscope. The number of vesicles in cubic mm area is calculated using the...