Drug Content in the Nanomicelles

In order to evaluate their drug content, the nanomicelle dispersions were diluted (1:3 v:v) with an ethanol:water (1:1, v:v) mixture and analyzed by UV spectroscopy (Shimadzu UV-1601) at 360 nm for fenretinide and 250 nm for lenalidomide, in comparison with the empty nanomicelles. The concentrations obtained represented the drugs encapsulated in the nanomicelles and free in the aqueous phase. Therefore, to obtain the concentrations of free drugs, the nanomicelle dispersions were centrifuged in a 3.5 mL Ultra 5 KDa filter (Merck Millipore) at 4000 x g for 30 min, and the ultrafiltrate was spectrophotometrically analyzed for drug content, after dilution 1:3 with an ethanol:water (1:1, v:v) mixture. The difference between the whole drug concentration in the nanomicelle suspension and in the ultrafiltrate provided the concentration of the encapsulated drugs. The drug loading was obtained as the ratio between the concentration (w:v) of the encapsulated drug and the concentration (w:v) of the nanomicelle dispersion. The encapsulation efficiency as the ratio between the concentration (w:v) of the encapsulated drug and the whole drug concentration (w:v) obtained by spectrophotometric evaluation of the liquid dispersion before purification by SEC.