Biuret

For preparation of nanoparticles, procedure described in Kondapi et al., [49] was adopted. 25 mg of apotransferrin dissolved in 100 µl of phosphate buffer saline (PBS) or dimethyl sulphoxide (DMSO). The solution of apotransferrin (100 µl) was slowly mixed with a 100 mM of doxorubicin hydrochloride (3.46 mg in 100 µl DMSO) and the mixture was incubated on ice for 5 min. The mixture of apotransferrin and the drug was slowly added to 30 ml of olive oil at 4°C with continuous dispersion by gentle vortexing. The sample was sonicated at 4°C using an MSE sonicator probe (PG43301, MSE Instruments, UK) or equivalent with a 30 sec period pulse, having an amplitude of 5 µm. This sonication step was repeated 15 times with a gap of 1 minute between successive steps. The resulting mixture was immediately frozen in liquid nitrogen for 10 min. and was then transferred to ice and incubated for 4 hours. The particles formed were pelleted by centrifugation at 2915 x g for 10 min at 4°C and the pellet was extensively washed with diethyl ether and dispersed in phosphate buffered saline (PBS). The particles were then estimated for protein using Biuret method and the nanoparticles were expressed in terms of protein concentration equivalents. The possibility of protein or doxorubicin entrapment in the form of emulsion along with the nanoparticles was assessed through the study of solubilization of the final pellet for water or DMSO soluble doxorubicin by fluorimetry and apotransferrin by Biuret method. No significant soluble forms of doxorubicin or apotransferrin were found thus ruling out the possibility of any emulsion formation during the particle preparation. Nanoparticles in pellet form were stable. When dispersed in PBS they were stable for 1 week at room temperature and for 2 months at 4°C and 6 months at −80°C.