Laborota 4003

The concentrate from nanofiltration was further processed to a dry powder by spray-drying (Buchi Mini Spray Dryer Model 191, Buchi Labortechnik AG, Flawil, Switzerland). Initially, the concentrate was partly evaporated (rotary evaporator Heidolf, Laborota 4003, Heidolph Instruments GMBh & CO KG, Schwabach, Germany) to form a solution of 10% TDS. Preliminary experiments showed that direct drying of the concentrate was not possible because particles were sticking to the drying chamber. Therefore, maltodextrin DE₁₀ was added to the solution at a final concentration of 13% (w/v) as a drying agent. The main constraint in choosing the operating conditions in this case was the outlet temperature of the product, which should be kept as low as possible to avoid degradation of the heat-sensitive polyphenols. Based on preliminary test runs, the chosen operating conditions were: drying air temperature, 140 °C; compressed air flow 0.7 m³/h; sample feed rate, 0.82 ± 0.05 g/min; and drying air rate, 21 m³/h. The collected powder was sealed in a vacuum package and stored at −20 °C until analysis for its antioxidant properties.

Two different compositions of liposomes were studied: formulation 1, using DOTAP, Mal-PEG and Chol, and formulation 2, using DOTAP, PC, Mal-PEG and Chol.
Both compositions were prepared using the classic thin-film method [23 (link)]. Briefly, lipids were dissolved in methanol as organic solvent. Methanol was then removed by rotary evaporation (Laborota 4003, Heidolph Instruments, Schwabach, Germany) at 38 °C under vacuum to avoid further toxicity. After 30 min, a thin lipid film was obtained. To remove the residual solvent, lipid film was dried under a stream of nitrogen for 2 h at room temperature. The film was then hydrated with a 5% v/v dextrose solution in water for formulation 1 or a 0.9% v/v sodium chloride solution in water for formulation 2, and then multilamellar vesicle (MLV) liposomes were obtained. Extrusion step was performed to reduce and homogenize liposomes in size through two 0.1 µm and two 0.08 µm polycarbonate pore membranes (Nucleopore, Whatman, Maidstone, UK) using LipoFast LF-50, and then small unilamellar vesicle (SUV) liposomes were obtained [24 (link)].
For each formulation, liposomes (i.e., ASO-Li-1 for formulation 1 and ASO-Li-1 for formulation 2) and immunoliposomes (i.e., ASO-iLi-1 and ASO-iLi-2) were generated.

A mixture of CUR (368 mg, 1 mmol), MPA (320 mg, 1 mmol), and DMAP (24 mg, 0.2 mmol) was dissolved in acetone (10 mL) to get a clear solution. The mixture was then added dropwise with a solution of EDC (287.55 mg, 1.5 mmol) in acetone (5 mL). The reaction mixture was stirred on an ice bath and kept at the temperature range between 0 and 5 °C for 30 min. To the reaction, 0.1 N HCl (10 mL) was added and stirred for 1 min. The reaction mixture was extracted with dichloromethane (3 × 30 mL). The combined dichloromethane extract was washed with DI water (2 × 20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure (Laborota 4003, Heidolph, Schwabach, Germany) to obtain a crude product in yellow (107 mg, 16% yield). The crude product was subjected to column chromatographic purification on a C18 column (40–63 µ, SiliCycle Inc., Quebec City, Quebec, Canada) using a mixture of methanol and water (8:2) as a mobile phase. Each eluent fraction was monitored by thin-layer chromatography (TLC) on a silica gel 60 F₂₅₄ plate (0.25 mm thickness) using a mixture of dichloromethane and methanol (15:1) as a developing solvent.