# Neuroprotective Investigation of Chitosan Nanoparticles for Dopamine Delivery
## 2 Materials and Methods
### Biological Studies  
Incubation cell conditionsHuman neuroblastoma SH-SY5Y cells were grown at 37 \({}^{\circ}\)C in a humidified incubator with 5% CO\({}_{2}\) in Dulbecco's modified Eagle's medium (DMEM)-F12 medium, which was supplemented with 10% newborn calf serum, 100 Units/mL penicillin and 100 mg/mL streptomycin.  
MTT test on SH-SY5Y cell lineAn MTT assay was used to determine the cell viability in different conditions. For this, cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin in a humidified atmosphere of 5% CO\({}_{2}\) at 37 \({}^{\circ}\)C. These were plated at 5 \(\times\) 10\({}^{3}\) cells per well in a 96-well plate. After treatment, the culture medium was aspirated, before 100 mL of RPMI-phenol free medium containing 10 mL of MTT stock solution and 5 mg/mL of PBS solution was added to each well. After one hour of incubation, the MTT solution was removed and 100 mL of DMSO was added to dissolve MTT-formazan crystals. The absorbance of the converted dye was measured at a wavelength of 570 nm using an iMark microplate reader (Bio-Rad, Hercules, CA, USA). The relative cell viability was expressed as a percentage of the untreated control group.  
Determination of intracellular reactive oxygen speciesHydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) was measured by Amplex\({}^{\circledR}\) Red Hydrogen Peroxide/Peroxidase Assay Kit (TermoFisher Scientific,Waltham, MA, USA). The oxidation product of 1:1 stoichiometric reaction between Amplex(r) Red reagent and H\({}_{2}\)O\({}_{2}\) was spectrophotometrically measured at 571 nm.  
_Glutathione peroxidase activity assay._ The glutathione peroxidase (GPx) assay was performed on control cells and on cells incubated with 100 uM DA, 100 uM CS(DA) NPs and the equivalent amount of CS NPs for 24 h following the instructions reported in [PERSON]'s GPx assay kit (Kayman Chemical, Ann Arbor, MI, USA). The activity was measured indirectly by a coupled reaction with glutathione reductase. Oxidized glutathione (GSSG) produced upon reduction of hydroperoxide by GPx is recycled to its reduced state by glutathione reductase (GR) and nicotinamide adenine dinucleotide phosphate (NADPH). The change in absorbance at 340 nm was monitored for 3 min. A blank with all ingredients except the sample was also monitored. Specific activity was calculated as U/mg protein.  
_Superoxide dismutase activity assay._ An enzymatic assay was performed on control cells and on cells incubated with 100 uM DA, 100 uM CS(DA) NPs and the equivalent amount of CS NPs for 24 h. Superoxide dismutase (SOD) activity was assayed with [PERSON]'s assay kit, which utilized a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. SOD activity was calculated using the equation obtained from the linear regression of a standard curve built in the same experimental conditions.  
_Statistical analysis._ The analytical data were expressed as mean \(\pm\) standard deviation. Statistical analysis was conducted using Student's \(t\)-test or one-way analysis of variance (ANOVA). Levels of statistical significance were set at \(p<\) 0.05, 0.005 and 0.001, as stated in the text.