2.5. Drug Encapsulation Efficiency (EE) and Drug Loading (DL)

The encapsulation efficiency (EE) and drug loading (DL) of DOX in NLC were determined by an ultrafiltration method, using centrifugal devices (Amicon^® Ultra−0.5 mL 100 k; Millipore, USA). To eliminate the binding of DOX to the devices, a pretreating of the filters was performed as previously described [45]. The devices were soaked in a passivating solution (Tween 20 ^®, 5% w/v), maintained overnight at room temperature and washed with distilled water prior to use. The EE and DL were calculated by using the following Equations (1) and (2), respectively:

where CT = total DOX concentration in NLC; CAP = DOX concentration in aqueous phase (non-encapsulated drug); WDL = mg of drug loaded in NLC and WNP = g of lipids.

The CT, CAP and WDL were evaluated as described by Mussi et al. [27]. The CT was analyzed by dissolving an aliquot of the NLC dispersion in a mixture of tetrahydrofuran (THF)/methanol (MeOH) 40:60 v/v, followed by centrifugation (10 min at 2400 g) and analysis of the supernatant by UV-vis spectrophotometry at 480 nm (UV-Vis Evolution 201; ThermoFisher, Shanghai, China). The CAP was evaluated from an aliquot of the aqueous phase separated from the NLC dispersion by ultrafiltration (10 min at 2400 g), dilution with THF/MeOH and analysis by UV-vis. The WDL was derived by using the calculated EE × total (mg) of DOX added. The method for DOX quantification was previously validated. The five-point linear regression analysis resulted in the following linear equation: y = 0.01930X–0.00615 (R² = 0.9915).