Amicon ultra centrifugal filters ultracell 3 kda

The NC-loaded NLCs were diluted in ultrapure water and then filtered (Amicon^® Ultra Centrifugal Filters Ultracell^®—3 kDa, Merck Millipore Ltd., Tullagreen, Carrigtwohill; from Sigma-Aldrich Química, S.A), at 14,500× g and 25 °C for 3 min to separate the free compounds from the compound-loaded NLCs. A mixture of 3:7 (v/v) of the supernatant and pure ethanol was prepared and the amount of free compound was quantified by UV-Vis absorbance measurements (BioTek Synergy HT Microplate Reader, BioTek, Winuschi, VT, USA) at the characteristic wavelength of each compound (282 nm for NDGA, 289 nm for naringenin, and 367 nm for kaempferol). The amount of the free compound was calculated through a calibration curve for each compound solution. This determination was performed in triplicate. Encapsulation efficiency (EE) and loading capacity (LC) were calculated through Equations (1) and (2): $$\mathrm{EE} (\%)=\frac{\mathrm{Total} \mathrm{amount} \mathrm{of} \mathrm{added} \mathrm{NC}-\mathrm{Unentrapped} \mathrm{NC}}{\mathrm{Total} \mathrm{amount} \mathrm{of} \mathrm{added} \mathrm{NC}}\times 100$$
$$\mathrm{LC} (\%)=\frac{\mathrm{Total} \mathrm{mass} \mathrm{of} \mathrm{encapsulated} \mathrm{NC}}{\mathrm{Total} \mathrm{mass} \mathrm{of} \mathrm{NLC}+\mathrm{Total} \mathrm{mass} \mathrm{of} \mathrm{encapsulated} \mathrm{NC}}\times 100$$

The in vitro release profiles of the NC-loaded NLCs were assessed in simulated physiological environment. The NC-loaded NLCs were diluted in a 10 mM solution of HEPES hemisodium salt buffer to a volume of 5 mL and placed at 37 °C and continuous stirring of 200 rpm. At predetermined timepoints, aliquots of 200 µL were collected from the release medium. To separate the released compounds from the NLCs, the samples were ultra-centrifugated (Amicon^® Ultra Centrifugal Filters Ultracell^®—3 kDa, Merck Millipore Ltd., Tullagreen, Carrigtwohill; from Sigma-Aldrich Química, S.A) at 14,500× g and 25 °C for 3 min. The amount of the released compound was quantified by UV-Vis absorbance as described in Section 2.2.4 (BioTek Synergy HT Microplate Reader, BioTek, Winuschi, VT, USA). This test was performed in triplicate. The percentage of the released natural compound (NC) at each time point was calculated from the following equation: $$\mathrm{Released} \mathrm{NC} (\%)=\frac{\mathrm{Amount} \mathrm{of} \mathrm{NC} \mathrm{at} \mathrm{time} \mathrm{t}}{\mathrm{Total} \mathrm{amount} \mathrm{of} \mathrm{NC}}\times 100$$