In vitro drug release studies

Studies were performed in simulated intestinal fluid (SIF: pH 7.4), for a period of 24 h.^16,17,18 A dialysis (cellulose membrane) method was used to identify release behavior of nanoparticles. 5 mg nanoparticle formulation was weighed into the dialysis bag with 1 mL of dissolution medium. The dialysis bag which was sealed with clamps was placed in 50 mL of the SIF at 37±0.5°C under sink conditions and stirred at 100 rpm using magnetic stirrer. In order to determine the amount of DL released from the dialysis bag at different time intervals (5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 5 h 6 h, 24 h), 1 mL of the samples were picked up and then replaced with the 1 mL of fresh SIF. The concentration of drug released to the medium was determined by measuring the absorbance at 262 nm using HPLC Shimadzu liquid chromatography equipped with a model LC-10ATVP binary pump and model SPD-M10AVP PDA detector using the stationary phase 150x4.6 mm LiChrospher^® 100 RP-18 octadecyl silane column (5 µm particle size) (Merck, Darmstadt, Germany) with integration by LC Solution Version 1.23 SP1 Software (Shimadzu Corporation, Kyoto, Japan). The mobile phase consisted of acetonitrile:water (60:40). The mobile phase was prepared daily and degassed by sonication under reduced pressure and filtered through 0.45 µm membrane filter. The flow rate was set at 0.8 mL/min resulting in a run time of 7 min per sample. The injection volume was 20 µL. Detection was performed at 262 nm and samples were analyzed at room temperature. In order to study the mechanism of drug release from nanoparticles, the release data were fitted to different equations with DDSolver programme.