3.4. Encapsulation Efficiency (EE) and Drug-Loading Capacity (LC) Estimations

The encapsulation efficiency and loading capacity of S14-loaded NPs were determined by a direct method with HPLC analysis [28]. The samples were analyzed in a SunFire^® C18, 3.5 μm, 4.6 × 50 mm² column (Waters, Cerdanyola del Vallés, Spain) and UV-Vis spectra were acquired using a Thermo Finnigan Surveyor UV-Vis Plus Detector (ThermoFisher, Madrid, Spain). The entrapped drug (S14_entrapped) was detected by direct injection of the samples after breaking the nanoparticles as follows: 2.5 mg of NPs were dissolved in 5 mL of acetonitrile and the mixture was sonicated for 25 min. Then, 1 mL of solution was filtered over a 0.22 μm filter (Minisart^® Syringe Filter, Polietersulfona (PES), ethylene oxide, Cat.#16532-K) and analyzed in the HPLC system. The encapsulation efficiency and drug-loading capacity values were expressed according to the following Equations:

For the calibration curve, six standard solutions at concentrations ranging from 0.2 mM to 0.7 mM of free S14 were prepared and analyzed under the same conditions.