For the investigation of IFN-β decomposition kinetics 52.3 μg ml−1 IFN-β solution was prepared in PBS, and the protein concentration was measured for 4 days using the mBCA assay. The absorbance due to the colour reaction was spectrophotometrically analysed at 562 nm in predetermined times.
The prepared BSA-loaded nanoparticles were washed and redispersed in PBS containing 0.03% w/v sodium azide to get a nanoparticle concentration 5–6 mg ml−1. The washed IFN-β-loaded nanoparticles were suspended in human blood plasma (1 mg ml−1). The release medium was replaced after sampling. The samples were mixed by a rotating mixer (Bio RS-24 mini-Rotator, Biosan, Latvia) with vertical rotation at 30 rpm, and incubated at 37 °C in a G24 Environmental Incubator Shaker (New Brunswick Scientific, USA). At predetermined intervals, 0.5 ml of each sample was ultracentrifuged (Beckman Optima Max-E) for 10 min at 40 000×g. The concentration of released IFN-β was determined using ELISA. The BSA concentration was followed by mBCA assay in the supernatant after removal of the nanoparticles by centrifugation, and the IFN-β was analysed in the removed supernatants by ELISA using a multiplate reader.
The prepared BSA-loaded nanoparticles were washed and redispersed in PBS containing 0.03% w/v sodium azide to get a nanoparticle concentration 5–6 mg ml−1. The washed IFN-β-loaded nanoparticles were suspended in human blood plasma (1 mg ml−1). The release medium was replaced after sampling. The samples were mixed by a rotating mixer (Bio RS-24 mini-Rotator, Biosan, Latvia) with vertical rotation at 30 rpm, and incubated at 37 °C in a G24 Environmental Incubator Shaker (New Brunswick Scientific, USA). At predetermined intervals, 0.5 ml of each sample was ultracentrifuged (Beckman Optima Max-E) for 10 min at 40 000×g. The concentration of released IFN-β was determined using ELISA. The BSA concentration was followed by mBCA assay in the supernatant after removal of the nanoparticles by centrifugation, and the IFN-β was analysed in the removed supernatants by ELISA using a multiplate reader.