# Neuroprotective Investigation of Chitosan Nanoparticles for Dopamine Delivery
## 3 Results
### ROS Neuroprotection by CS(DA) NPs and Enzymatic Activities  
It is reported that when DA exists outside of synaptic vesicles at high concentrations, it can induce oxidative stress in cells [24; 25]. This is because free DA is an unstable molecule that can oxidize spontaneously to form ROS, free radicals and quinones. For this reason, synaptic vesicles play a critical role in storing and thus, preventing DA oxidation in dopaminergic neurons. Moreover, DA is also easily metabolized by monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetic acid with the concomitant formation of hydrogen peroxide [24]. DA or CS(DA) NPs were co-cultured with dopaminergic SH-SY5Y cells at suitable 5% CO\({}_{2}\)-humidified atmosphere for 24 h. The percentage of cell viability was first evaluated by optical microscopy, before being evaluated by an MTT assay. We performed a dose dependent (25-100 uM) study for both free DA and CS(DA) NPs. The effects observed in cells treated with CS(DA) NPs were compared with those obtained from both CS NPs treated and control cells (treated with vehicle alone). In viable cells, MTT is cleaved by mitochondrial dehydrogenase to produce a purple product, which has a concentration (measured in terms of absorbance) that is directly proportional to the number of viable cells and is inversely proportional to the degree of cytotoxicity. The addition of free DA (25-100 uM) to cells cultured in the DMEM-F12 medium showed no obvious toxicity with respect to control cells, which was demonstrated by both optical microscopy and MTT analysis (Figure 3). On the other hand, the partial loss of cell viability was induced by CS(DA) NPs at all the investigated concentrations, with respect to both control and NPs alone. However, the cell viability was always above 75% (Figure 3b). A further increase in the concentration of free DA caused more significant cellular death, which was up to 70% with 250 uM of free DA (data not shown). This is why we decided to investigate a significantly milder concentration range up to 100 uM, thus resembling more normal physiological conditions of the cellular environment. Figure 2: In vitro DA release profiles from CS(DA) NPs in PBS solutions at a pH of 7.4 (blue dots) and a pH of 5.5 (orange dots) at 37 \({}^{\circ}\)C. Error bars represent the standard deviation values calculated from three independent experiments. In order to obtain a better understanding of the neuroprotective effect exerted by the chitosan polymer over the delivery of free DA, we decided to inspect the generation of oxygen radicals in different conditions, which involves namely using polymeric chitosan NPs, free DA and CS(DA) NPs. As already stated, we chose to treat cells with free DA at a maximum concentration of 100 \(\upmu\)M to simulate a condition in which cells are not affected by external DA administration and are supposed to exert their normal physiological activity. Incubation of cells with free DA and CS(DA) NPs for 24 h caused a significant increase (\(p<0.001\)) in oxidative stress with respect to the control at 100 \(\upmu\)M concentration, which was indicated by increased H\({}_{2}\)O\({}_{2}\) production (Figure 4). It is worth noting that the addition of CS NPs at the same polymeric concentration used with 100 \(\upmu\)M CS(DA) NPs did not cause any significant toxicity to the cells (Figure 3b). However, oxidative stress in cells treated with CS(DA) NPs at the same concentration was significantly lower with respect to the values measured in cells treated with DA alone. This is probably due to the sustained release of DA over time, which is exerted by the nanoparticles, compared to the addition of 100 \(\upmu\)M free DA in one single injection. Intracellular mechanisms of protection from oxidative free radical damage include scavenging enzyme systems, which reduce the amount of ROS, such as superoxide dismutase (SOD) and glutathione peroxidase (GPx).