2.3. Dry Powder Preparation by Spray-Freeze-Drying (SFD)

A total of 16 formulations were prepared, 11 according to the factorial design plus five extended formulations (Table 2). The feed solutions for SFD were prepared by mixing appropriate volumes of BSA and HPβCD stock solutions with ultrapure water to achieve a total solute mass of 120 mg. Stock solutions of BSA and HPβCD for the factorial formulations were prepared at 15 mg/mL and 150 mg/mL, respectively, while stock solutions of BSA and HPβCD for the extended formulations were prepared at 100 mg/mL. For the SFD step, the feed solution was first drawn into a 10 mL syringe (Terumo Corporation, Tokyo, Japan), which was connected via a tube to a two-fluid nozzle (Büchi Labortechnik AG, Flawil, Switzerland) of 0.7 mm internal diameter for atomization. The nozzle was positioned above a stainless-steel tank containing liquid nitrogen to facilitate instantaneous freezing [7]. The nitrogen gas flow rate for atomization was set according to the factorial design. The feed solution was then fed into the nozzle at a controlled feed rate of 2 mL/min using a syringe pump (Legato^™ 210, KD Scientific, Holliston, MA, USA). The atomized liquid droplets were immediately frozen as they traveled towards the liquid nitrogen. Primary drying was conducted in a freeze-dryer (FreeZone^® 6 Liter benchtop freeze-dry system with stoppering tray dryer, Labconco Corporation, Missouri, MO, USA) at a chamber pressure below 0.14 mbar at −25 °C for 20 h. Following this, the temperature was gradually increased to 20 °C over 4 h and thereafter kept constant for at least another 40 h to allow secondary drying. The dried powders were collected and stored in a desiccator with silica gel (10% humidity as monitored) at ambient temperature until further analysis. The production yield was calculated as the percentage of the mass of powder collected to the initial solute mass input, assuming negligible moisture content in the collected powder.

Formulations of the spray-freeze-dried powders. CP: center point; EXT: extended formulation; an apostrophe denotes high-level factor.