# Physicochemical Characterization of Hyaluronic Acid and Chitosan Liposome Coatings
## 3 Results
### Physicochemical Properties of the Coated Liposomes
#### 3.1.1 Size Distribution and \(\zeta\)-Potential  
The results obtained from the physicochemical characterization of the coated and uncoated liposomes are reported in Table 1. The DLS experiments were performed after the purification processes.  
The first step was to analyze the size dimensions of DOPC/DOPE zwitterionic liposomes with any surface modification to understand the behavior of CH and HA during the coating processes. The zwitterionic vesicles showed a hydrodynamic ratio that was in agreement with the extrusion procedure. The dispersions showed average hydrodynamic diameters typically obtained for large unilamellar vesicles, since the values are greater than 100 nm [29]. The PDI values suggested that the liposome solution showed the properties of a pure monodisperse system. The surface charge of 25.5 \(\pm\) 9.4 mV indicated that such systems are quite stable in solution.  
The size of the zwitterionic HA-coated liposomes revealed higher values with respect to the uncoated derivatives (264.4 \(\pm\) 12.5 nm). These liposomes showed an average diameter that was in perfect agreement with the preparation method, and the PDI value suggested the presence of monodisperse systems. The coating of the liposome with HA deeply modified the surface charge, and the \(\zeta\)-potential value showed that aggregation/flocculation  
\begin{table}
\begin{tabular}{c c c c} \hline
**Liposome** & **Mean Diameter \(\pm\)** & **Polydispersity Index** & \(\boldsymbol{\zeta}\)**-Potential \(\pm\)**esd** \\
**Composition** & **esd (nm)** & **(PDD)** & **(mV)** \\ \hline DOPC/DOPE & 160.5 \(\pm\) 3.3 \({}^{\text{a}}\) & 0.15 & 25.5 \(\pm\) 9.4 \({}^{\text{a}}\) \\ \hline DOPC/DOPE-HA & 264.4 \(\pm\) 12.5 \({}^{\text{b}}\) & 0.20 & 4.9 \(\pm\) 3.3 \({}^{\text{b}}\) \\ \hline DOPC/DOPE-CH & 450.3 \(\pm\) 16.7 \({}^{\text{c}}\) & 0.35 & 55.3 \(\pm\) 8.2 \({}^{\text{c}}\) \\ \hline \end{tabular}
\end{table}
Table 1: Size distribution with polydispersity index (PDI) and \(\zeta\)-potential for all synthesized liposomes. The values are the average of three measurements (mean \(\pm\) esd) and different letters in the same column indicate significant differences (_p_ < 0.05, Tukey’s test).  
processes in solution are very likely. The aggregates can increase their size as a function of storage time.  
CH-coated liposomes showed much higher size values with respect to uncoated derivatives (450.3 \(\pm\) 16.7 nm), with PDI values suggesting a polydisperse system. The \(\zeta\)-potential value confirmed the strong stability of the suspensions versus time. The values also suggested that the coating process properly occurred, resulting in systems with a higher average diameter than the uncoated liposomes. The coating therefore influences the size of the vesicles, improving their stability and thus preventing aggregation in solution. The higher values obtained could be considered as indicative of excellent stability, since the line between stable and unstable dispersions is generally set at either +30 or \(-\)30 mV [30].  
DLS experiments highlighted that the liposomes coated with CH had a higher mean diameter than those coated with HA, confirming the presence of CH on the surface of vesicles. The \(\zeta\)-potential values also confirmed the presence of either of the two polymers on the liposome surfaces.