0.2 μm cellulose membrane syringe filter

Radical scavenging activity was measured using the DPPH (2,2-diphenyl-1-picryllhydrazyl) radical scavenging method described by Brand-Williams et al. [29 (link)] with modifications. Briefly, Se-loaded nanoparticles (5 mg Se based on entrapment efficiency results) and equivalent mass of unloaded nanoparticles were suspended in 5 mL of deionized (DI) water and stirred at 100 rpm in the dark for 48 h at 25°C. Next, samples were filtered using 0.2-μm cellulose membrane syringe filter (MilliporeSigma), and 0.1 mL-aliquot of each sample was vigorously mixed with 3.9 mL of 0.01 mmol DPPH radical in methanol. Then, each solution was incubated for 30 min in the dark at 25°C and their absorbances were measured at 517 nm (Synergy H1 Hybrid Multi-Mode Reader). The radical scavenging activity, expressed in % scavenging ratio (I) was calculated using Eq 2:
$\mathit{I}=\left[\frac{\left({\mathit{A}}_{\mathit{o}}-{\mathit{A}}_{\mathit{i}}\right)}{{\mathit{A}}_{\mathit{o}}}\right]\mathit{x}\mathbf{100}$
Where A_o and A_i are the absorbances of the blank (0.1 mL DI water + 3.9 mL 0.01 mmol DPPH) and nanoparticle samples, respectively.

Selenium release from chitosan nanoparticles was evaluated by suspending them in phosphate buffer saline (PBS, pH 7.4) at 1 mg mL^-1 of Se based on entrapment efficiency results. Samples were divided in 5-mL aliquots and transferred to a shaker under constant agitation at 100 rpm and 28°C in the dark. The temperature was selected to simulate the conditions observed during Nile tilapia cultivation. At incremental time intervals up to 48 h the Se released was measured. At predetermined time intervals, a sample was filtered using 0.2-μm cellulose membrane syringe filter (MilliporeSigma) and then Se content was measured using the method described in the Entrapment efficiency section.