Ultrafree centrifugal amicon ultra 4 centrifugal filter devices

The drug encapsulation efficiency (EE) of nanoparticles was evaluated by measuring the absorption at the wavelength of 319 nm using ultraviolet (UV)/visible (VIS) spectrophotometry (Jasco, Easton, MD, USA). The lyophilized formulation samples with drug were diluted in double-deionized water (1:200) and centrifuged. The pellet was dissolved in 400 μL of acetonitrile and vigorously vortexed for 30 minutes to completely extract the drug from the nanoparticles. Then, the resulting solution was transferred into Ultrafree^® Centrifugal Amicon^® Ultra-4 Centrifugal Filter Devices, with nominal molecular weight cutoff 10,000 kDa MWCO (Millipore, Billerica, MA, USA). Centrifugation was carried out using a Jouan BR4i multifunction centrifuge with a KeyWrite-D™ interface (Thermo Electron, Waltham, MA, USA) with a fixed 23°-angle rotor and 3,000× g spin for 8 minutes at 20°C. The drug released from the nanoparticles was present in the supernatant, which was stored in the centrifuge tube until quantification by UV/Vis spectrophotometry. The EE of RFB was defined as the ratio between the quantity of RFB measured in the supernatant and initial quantity of drug added to the formulation.15 (link)
$\text{EE}=\frac{\text{Total amount of RFB in nanoparticles}}{\text{Initial amount of RFB}}\times 100$

The EGCG, EE, and LC of the lipid NPs were evaluated by measuring absorption at the wavelength of 273 nm using a UV/Vis spectrophotometry (Jasco, Easton, MD, USA). The lipid NPs with EGCG were diluted in double-deionized water (1:40) and centrifuged. To separate the lipid NPs from their aqueous medium, the diluted samples were centrifuged in Ultrafree^® Centrifugal Amicon^® Ultra-4 Centrifugal Filter Devices, with nominal molecular weight cutoff 50,000 kDa molecular weight cut-off (MWCO) (Millipore, Billerica, MA, USA) at 25°C for 10 minutes or until complete separation. The supernatant with the dissolved EGCG was collected and measured. The EE of EGCG was defined by the ratio of the amount of EGCG measured (in mg) in the supernatant to the initial amount of EGCG added (in mg) to the formulation, while the LC of EGCG was determined by calculating the ratio between the amount of EGCG measured in the NPs and the total amount of EGCG and lipids present in the formulations.11 (link)
$\begin{array}{l}\text{EE}=\frac{\text{Total amount of EGCG}-\text{unentrapped EGCG}}{\text{Total amount of EGCG}}\times 100\\ \text{LC}=\frac{\text{Total amount of EGCG}-\text{unentrapped EGCG}}{\text{Total amount of lippids}+\text{tensioactive}+\text{EGCG}}\times 100\end{array}$