# Neuroprotective Investigation of Chitosan Nanoparticles for Dopamine Delivery
## 3 Results  
### Preparation and Characterization of the NPs  
The preparation of the chitosan NPs was accomplished using the gelation method with TPP as the chelating agent according to previously reported procedures [16; 22]. Briefly, the CS NPs were prepared by dissolving the desired amount of DA (5 mg/mL) in the starting chitosan solution, before TPP was added in order to allow the encapsulation of the antioxidant. Thus, we finally obtained CS(DA) NPs. Bare CS NPs were also prepared and used as control in the experiments with cells. The shape and the morphology of the CS(DA) NPs were observed by transmission electron microscopy (TEM; Figure 1). The images revealed that all NPs were quite spherical and uniform in shape. The observed mean diameter was about 80 nm for all the formulations, although some variability was clearly visible with sizes in the range of 50-100 nm. No aggregation could be observed among the NPs. Figure 1: TEM image of CS(DA) NPs. The hydrodynamic diameter in the PBS solution (pH of 7.4) and the corresponding polydispersity index (PDI) of the NPs were observed by DLS and the results are reported in Table 1. The formulations showed an average diameter of about 100 and 109 nm for the CS and the CS(DA) NPs, respectively. As expected, the measured size in the solution was slightly higher compared to that observed by TEM. The observed charge of the NPs in solution was approximately 27 and 34 mV for the CS and CS(DA) NPs, respectively (Table 1). These positive values are consistent with previously reported values in the literature and are due to the free amino groups exposed to the outer environment. As expected, a bigger size of NPs results in a larger positive net charge. The DA loading was indirectly calculated by measuring the absorbance of the starting solution used during the synthesis and the absorbance of the same solution after the gelation process. The observed DA concentration in the solution was subtracted from the starting concentration and the resulting value was assumed to be the amount of DA encapsulated into the NPs. An average encapsulation efficiency of 64% was observed over five syntheses, although this value was quite variable with the efficiency in the range of 45-75%. Furthermore, we were not able to gain perfect control over this parameter. In order to reduce variability in the subsequent experiments, two stock solutions of several milliliters each were prepared for the CS and CS(DA) NPs and these were used in all the subsequent characterization and cellular experiments. To better understand the neuroprotective effect exerted by the CS(DA) NPs, the drug release was investigated in vitro by mimicking the environmental conditions encountered in the cellular experiments. Two physiological conditions were investigated by means of PBS solutions. The first one was conducted at a pH of 7.4, which is similar to the environment found in the cellular medium. The second one was conducted at a pH of 5.5, mimicking the intracellular endosome and lysosome ambient pH encountered after internalization of the NPs [23]. In both conditions, most of the DA release was obtained in the first few hours, with saturation almost reached after approximately 24 h (Figure 2). The measured concentration of released DA in solution at a pH of 7.4 reached almost 50% of the initial encapsulated concentration after 48 h. As expected, the acidic conditions allowed a slightly faster DA release compared to that observed at an almost neutral pH, while the percentage of drug released was also slightly higher after 2 days. This was probably due to the protonation of the amine groups at the acidic pH, which allowed a double effect. On one hand, the electrostatic repulsion among the positively charged amines of the chitosan induced swelling of the NPs. On the other hand, the electrostatic repulsion between the amine groups of the chitosan and that of the DA allowed slightly more efficient total release of the drug after 48 h.