Freezone 4.5 liter freeze dry system

The morphologies of the freeze-dried cellulose hydrogels were carried out using a Hitachi Tabletop Microscopes TM 4000 Plus at an accelerating voltage of 5 kV with a 5 nm gold coating (Quorum Q150T S Plus Sputter Coater). Before the observation in SEM, the hydrogel samples were swollen to equilibrium, and then immersed in liquid nitrogen. The frozen samples were lyophilized by the vacuum freeze drier (Labconco Freezone 4.5 Liter Freeze Dry System). In the process of gold coating, the sample is positioned in the sputter coater vacuum chamber where gold is sputtered onto its surface using argon plasma.

The adhesive mix comprised two components, the WPI solution and the GTA solution. WPI (protein 90.4%, and total solids 95.58%) was purchased from Fonterra Ltd. (Auckland, New Zealand). At refrigerated temperature, the WPI was rehydrated in sterilized deionized water (4 °C) at a concentration of 4%, and then filtered (4 °C) by a Millex™ syringe with sterile filter unit (pore size: 0.22 µm) (Millipore Corporation, Bedford, MA, USA) to remove bacterial cells. The filtrate was freeze dried by a FreeZone^® 4.5 Liter Freeze Dry Systems (model 7751020) (Labconco Corporation, Kansas, MO, USA). The whole process was conducted under sterile conditions to minimize contamination. The sterilized freeze-dried WPI powder was reconstituted using sterilized deionized water to make the WPI solution at concentrations of 30.0%, 35.0%, 40.0%, and 45.0% (w/w). GTA (50.0% solution, w/w) purchased from Fisher Scientific (Fair Lawn, NJ, USA) was diluted using sterilized deionized water to 6.0%, 8.0%, 10.0%, and 12.0% (w/w), respectively.

The preparation of polymeric nanoparticles was performed using the nanoprecipitation technique [16 (link)]. The acetone-based organic phase was tested in two different volumes, either 5 mL or 10 mL, in which RSV was solubilized in different amounts, 1, 5, or 10 mg. In this same phase, 10 mg of PCL polymer flakes were also dissolved. The aqueous phase (20 mL) was composed of both 0.15% (w/v) of Poloxamer 407 with or without TPGS solubilized in different ratios 0.005%; 0.01%; 0.015%; 0.03% and 0.05% (w/w) in relation to the amount of PCL in phosphate-buffered saline (PBS), pH 7.4. The next step of the technique was the slow dripping of the organic phase into the aqueous phase, which was constantly stirred at 200 rpm at 30 °C, overnight [17 (link)].
For the solid-state characterization of the nanoparticles and for the stability study, they were lyophilized using ratios of 10%, 20%, and 30% of sucrose as a cryoprotectant, in relation to the amount of PCL. Samples were frozen at −80 °C for 24 h and then lyophilized for 48 h (FreeZone 4.5 Liter Freeze Dry Systems-Labconco). Lyophilized samples were stored in sealed falcon tubes and conditioned in a refrigerator.

Harvested cells were pelleted by centrifugation at 3000 rpm for 5 min, frozen at − 80 °C for ~ 1 h and subsequently freeze-dried overnight using a FreeZone^® 4.5 Liter Freeze Dry Systems (Labconco Corporation, USA) to obtain the dry cell weight (DCW) of each culture. Dry cells (~ 20 mg) were treated with 2 mL methanol/hydrochloric acid/chloroform (10:1:1, v/v/v) and heated at 90 °C for 1 h in sealed test tubes to convert lipids to fatty acid methyl ester (FAME). FAME was washed with 0.9% NaCl solution (1 mL) and extracted with hexane after mixing. FAME samples (1 µL) were analyzed by Agilent 7890A gas chromatography with flame ionization detection (GC-FID) as described previously [11 ].