Drug Loading (DL) and Release

Curcumin-loaded SPNs (Curc-SPNs) were synthesized by employing the same emulsion/solvent evaporation technique described above. Simply, the 200 µg of Curcumin was dissolved in chloroform and added to the oil phase per each milligram of PLGA. To estimate loading and encapsulation efficiency (EE), SPNs were resuspended in 1 ml of water and freeze-dried. After lyophilization, a known amount of particles was dissolved in acetonitrile to free the entrapped drug. The absorbance of Curcumin at 430 nm, with a baseline wavelength of 650 nm, was measured and used to calculate the amount of molecule. Each sample was evaluated in triplicate. Loading efficiency was expressed as the weight percentage of drug mass with respect to the total mass of the nanoparticles, whereas EE was expressed as the weight percentage of entrapped drug mass as compared to the initial drug input. The in vitro release of Curcumin was evaluated under physiological conditions in PBS at 37°C and pH 7.4 up to 72 h. 200 µl of nanoparticle solution at 10 µM Curcumin were transferred into Slide-A-Lyzer MINI dialysis cups with a molecular cutoff of 10 kDa (Thermo Scientific, Rockford, IL, USA) and dialyzed against 4 l of PBS. At each time point, three replicates were retrieved and analyzed. Quantification of the amount of drug released was obtained through a spectrophotometric measurement, using a method akin to the one employed in assessing the loading efficiency. The content of each cup was collected and centrifuged at 12,000 rpm for 20 min, and the resulting pellet of nanoparticles was dissolved in acetonitrile. Then, for each sample, the absorbance at 430 nm with a baseline wavelength of 650 nm was measured. Results for each time point are expressed as a percentage with respect to the initial time point.