Determination of Entrapment Efficiency and Drug Loading

Entrapment efficiency (EE %) and drug loading were evaluated using an indirect and direct method, respectively. For the indirect method, the nanoparticle suspension was centrifuged at 10,000 × g for 15 min to obtain a pellet. This was washed three times to remove free drug adhering to the surface of NPs. The supernatant was suitably diluted and free drug was analyzed using a previously developed and validated HPLC method. The EE % was calculated using the following formula:

Where, Wtotal Rufi is the amount of Rufi used in the preparation of nanoparticle formulation and Wfree Rufi is the amount of Rufi in the supernatant. In case of the direct method for estimation of EE %, the nanosuspension formulation was washed with three times its volume of water and centrifuged at 10,000 × g. The supernatant was discarded and the pellet obtained was dissolved in a suitable solvent. Further, it was centrifuged at 10,000 × g and the supernatant was diluted and analyzed for Rufi using a previously developed and validated HPLC method (Dalvi et al., 2018).