Fd 1000

After cultivation, the cells were harvested by centrifugation (6,400 g × 15 min, 4°C) and washed thrice with distilled water. The microbial cells or porcine brain were lyophilized using an FD-1000 vacuum freeze dryer (EYELA, Japan) at −80°C for 2 days. Lipids were extracted with chloroform/methanol/1% KCl solution (1.1: 1.1: 1, by volume), followed by extraction with chloroform twice. All extract solutions were collected, evaporated to dryness, and dissolved in methanol. This fraction was analyzed by ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) and subjected to further experiments.

The IR spectra of the freeze-dried liposomal formulations were obtained using an FTIR spectrophotometer (FTI-8400S, Shimadzu, Tokyo, Japan) as below. In brief, the optimized liposomal dispersions were frozen in 5% dextrose (also acts as cryoprotectant) at −80 °C for 24 h in an ultra-low temperature freezer. Liposomal dispersions incubated in simulated media were ultracentrifuged (100,000× g for 60 min) and washed with 5% dextrose before freezing. The frozen liposomes were freeze-dried (FD-1000, EYELA, Tohoku, Japan) for 48 h.
Then, freeze-dried liposomes (2 mg) were blended with potassium bromide (spectroscopic grade) (100 mg), then compressed into disks by a hydraulic press before being scanned from 4000 to 500 cm⁻¹ at a resolution of 4 cm⁻¹. Data were analyzed using FTIR software (IRsolution version 1.10, Shimadzu, Tokyo, Japan).

The surface morphology of each membrane was observed through scanning electron microscopy (SEM) imaging to study the impact of various TA concentrations. The membrane surface morphology was observed using a Field-Emission Scanning Electron Microscopy (FE-SEM, JSF-7500F, Jeol Co., Ltd., Tokyo, Japan). Before the analysis, samples were freeze-dried (FD-1000, Eyela, Tokyo, Japan) overnight. The membrane sample was coated with an osmium coater (Neoc-STB, Meiwafosis Co., Ltd., Shinjuku, Japan) to form a conductive ultra-thin osmium layer on the sample to impose conductive property.

PT-NLC was fabricated using the hot melt emulsification and sonication methods. In each experiment, liquid (Capryol 90^®) and solid lipid (GMS) were mixed in a 75 °C water bath. Five milligrams of PT were added to the melted lipids and mixed. Heated Tween 80 and poloxamer 188 were added and homogenized at 15,000 rpm for 1 min. They were sonicated with 108 W of amplitude for 10 min. After the cooling, formulations were freeze-dried with or without mannitol by lyophilizer (FD-1000, EYELA, Tokyo, Japan). Blank-NLC was fabricated without PT.

Powdered zein (1.0 g) was accurately weighed and dissolved into 20 mL of 80% (volume fraction) aqueous ethanol to form a stock solution. The stock solution was diluted with 50 mL of purified water containing SC at concentrations of 0, 10, 15, 20 and 25 mg mL⁻¹ to obtain five zein-SC mass ratios (1:0, 1:0.5, 1:0.75, 1:1 and 1:1.25). The dispersions were prepared under continuous stirring (1000 r min⁻¹) with a magnetic stirrer (Ret basic, IKA-Works Inc., Wilmington, NC) at room temperature. The solvent was removed from each dispersion under reduced pressure (Rotavapor N-1100, EYELA, Tokyo, Japan) for 10 min at 45°C. The residues were then subjected to centrifugation at 1,788.8 ×g for 10 min to separate the small quantity of zein that formed larger aggregates. The final dispersions were stored at 4°C until required for particle size, zeta potential measurements and scanning electron microscopy analyses. To obtain solid powder samples, the dispersions were freeze-dried for 24 h (FD-1000, EYELA, Tokyo, Japan). The powders were stored at −20°C before testing.

F-GelMA was synthesized as previously described with minor modifications [39 (link)]. In brief, 10 g of fish gelatin was dissolved in 100 mL sodium carbonate-bicarbonate buffer (0.1 M, pH 9.0), and different volumes (1.2 mL, 1 mL, 0.8 mL, 0.4 mL, 0.2 mL) of methacrylic anhydride were added 6 times every 30 times and the pH was adjusted to 9. Methacrylic anhydride was added at a rate of 0.5 mL/min to the fish gelatin solution with stirring at 50 °C and reacted for 3 h. The mixture was diluted 5-fold with distilled water to stop the reaction, and dialyzed using dialysis tubing (MWCO 12,000–14,000, Thermo Fisher Scientific, Waltham, MA, USA) for 48 h at 50 °C to remove unreacted methacrylic anhydride and methacrylic acid byproducts. The solution was lyophilized (FD-1000; EYELA, Tokyo, Japan) to generate a white porous foam and stored at 4 °C until use. Lyophilized F-GelMA was dissolved in HEPES buffer containing 0.5% (w/v) Irgacure 2959 as a photosensitizer at 80 °C, and used to fabricate hydrogels.