The particles were then recovered by ultracentrifugation (19,975g, 30min, 4��C, Cientec CT-15000R centrifuge, Brazil) and washed twice with water to remove the surfactant. The nanoparticles were dispersed in the cryoprotectant sucrose (5%, w/v), and the resulting selleck catalog nanosuspension was subsequently cooled to ?18��C and freeze-dried (Terroni, Brazil).The mean particle size, size distribution, and polydispersity index were determined by dynamic light scattering (BIC 90 plus, Brookhaven Instruments Corp.). The analyses were performed at a scattering angle of 90�� and a temperature of 25��C. For each sample, the mean particle diameter, polydispersity, and standard deviation for ten determinations were calculated.The amount of RVT in the nanoparticles was determined indirectly.

The supernatant obtained from the ultracentrifugation process was diluted in the mobile phase (1:1000), filtered through a 0.22��m pore-size filter and analyzed by the HPLC method previously developed and validated. The drug concentration in the supernatant was obtained by comparing the concentration to a previously constructed analytical curve. The amount of RVT trapped in the nanoparticles was determined by subtracting the quantity in the supernatant from the total quantity used during the preparation. These analyses were performed in triplicate.2.7. Nanoparticles Applicability 2.7.1. Antioxidant Activity Assay The cation radical ABTS��+ was employed to measure the antioxidant activity of free RVT, RVT-loaded nanoparticles (PLA and PLA-PEG nanoparticles), and blank nanoparticles.

A mixture of ABTS (7mM) and potassium persulfate (2.45mM) was prepared and allowed to stand at room temperature [22, 23]. The ABTS��+ solution was diluted to an absorbance of 0.70 at 734nm in a 50mM phosphate buffer, pH 7.4. Blank nanoparticles and different concentrations of RVT (free or nanoencapsulated) ranging from 1 to 25��M were used. The compounds were incubated at 37��C under constant agitation and protected from light for 0, 24, 48, and 72h. After incubation at 37��C, aliquots with known concentrations were incubated for 30min with ABTS��+, and the absorbance was measured at 734nm. The control used during this assay was the buffer solution. The percentage of inhibition was calculated by%Inhibition=(Ac��At)Ac��100,(2)where Ac is the absorbance of control and At is the absorbance of test.

The concentration of RVT that resulted in 50% of inhibition of ABTS��+ (IC50) was calculated by the linear regression of the RVT concentration versus percentage of ABTS��+ inhibition curves.3. Results3.1. Method DevelopmentInitial runs were Brefeldin_A performed using a mobile phase mixture of methanol and acetonitrile based on existing methods for RVT quantification in plasma [24]. Various ratios in the isocratic mode were tested, some of which led to the presence of more than one peak (methanol:acetonitrile 3:1 and 1.5:1).