4.12. Nitric Oxide (NO) Assay

The anti-inflammatory effects of the MSN formulations were evaluated by measuring nitric oxide (NO) production in RAW 264.7 cells using the Griess reagent (Sigma Aldrich, St. Louis, MO, USA). RAW 264.7 cells (1 × 10⁵ cells per well) were seeded into a 96-well plate and incubated at 37 °C with 5% CO₂ for 24 h. The cells were then treated with lipopolysaccharide (LPS) at a concentration of 1 µg/mL to induce inflammation. Various treatments were applied, including free Cxb at 5 and 25 µM, and MSN formulations loaded with celecoxib (MSN-NH₂-Cxb, MSN-NH₂-Cxb-PEI, and MSN-NH₂-Cxb-IP) at 5 and 25 µM. Blank wells, containing cells but no treatment, were included as a control. After 24 h of incubation with the treatments, the culture supernatants were collected, and NO production was measured using the Griess reagent. Briefly, 100 µL of supernatant was mixed with 100 µL of Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in 2.5% phosphoric acid) in a 96-well plate. The mixture was incubated at room temperature for 10 min, and the absorbance was measured at 540 nm using a microplate reader. The experiments were performed in triplicate, and the results are expressed as mean ± SD