# A Study on the Stoichiometry of Casein/Chitosan Gel Complexes as a Delivery System for Quercetin
## 3 Results and Discussion
### Characterization of Quercetin-Loaded Casein/chitosan Gel Polyelectrolyte Complexes
The values of the calculated parameters of the zero-order kinetics model are presented in Table 2.  
Based on the values of the release kinetic constant, the fastest release is observed in complexes S4 (with casein/chitosan charge ratio of 4:1), followed by samples S6 (with casein/chitosan charge ratio of 6:1). The slowest release rate is observed in samples S1 and S2, whose release kinetic constant values are practically identical. The main factors affecting the release rate are the diffusion coefficient of the drug through the carrier, the size of said carrier, and the physical state of the drug. The faster release from the S4 gel structures may be due to their smaller size and relatively smaller amount of chitosan in comparison to samples S1 and S2, which, in aqueous medium, swell greatly and prolong the diffusion path of the drug. As quercetin is a highly crystalline and poorly water-soluble compound, it is important to examine its phase state after it has been loaded in a drug delivery system. Differential scanning calorimetry and the X-ray powdered diffraction technique are employed to examine its crystallinity before and after the entrapment. The thermal stability and phase state of empty gel complexes, native quercetin, and quercetin-loaded gels have been investigated in the thermal region between room temperature and up to 350 \({}^{\circ}\)C (Figure 6). \begin{table}
\begin{tabular}{c c c c c} \hline
**Sample Name** & \(K_{0}\)**, [min\({}^{-1}\)]** & **DF Adj r\({}^{2}\)** & **Fit SE** & **F-Value** \\ \hline S1 & 0.0030 \(\pm\) 2.1\% & 0.986 & 0.054 & 758 \\ S2 & 0.0028 \(\pm\) 2.3\% & 0.984 & 0.054 & 301 \\ S4 & 0.0050 \(\pm\) 1.1\% & 0.996 & 0.046 & 3064 \\ S6 & 0.0036 \(\pm\) 1.6\% & 0.993 & 0.050 & 1512 \\ \hline \end{tabular}
\end{table} 
Table 2: Model parameters of the zero-order model describing the release kinetics of casein/chitosan complexes. The DSC curve of neat quercetin indicates two endothermic events corresponding to its dehydration temperature (130 \({}^{\circ}\)C) and melting point (328 \({}^{\circ}\)C), followed by rapid decomposition. These results are confirmed by other authors [44]. DSC thermograms of blank CAS:CH gel complexes show a wide endothermic peak related to water bound in the complexes [45], but no definite peaks due to melting or degradation. The sharp peak at 328 \({}^{\circ}\)C indicative of melting of native quercetin is absent from the thermogram of quercetin-loaded particles, while the characteristic peak at 130 \({}^{\circ}\)C is still visible, proving the immobilization of the drug in the complexes. According to [PERSON] et al. [46], this peak corresponds to the loss of endocrystalline solvent in three different polymorphs of quercetin. Transitions at temperatures around 100 \({}^{\circ}\)C are indicative of different thermal events, such as dehydration or glass transition, also mentioned as thermochemical transition. In the case of quercetin, the transition above 100 \({}^{\circ}\)C is a solid-solid thermochemical transition. Such a type of conversion is also reported for other flavanols and affects their ability to form hydrogen bonds. [PERSON] et al. [47] confirm this by pointing out the higher point of boiling water from the crystal lattice due to the strongly attached water molecules via hydrogen bonds to the quercetin crystals. Similar DSC thermograms were observed for all investigated samples. The crystallinity of quercetin was qualitatively confirmed by the X-ray powdered diffraction technique (Figure 7). Prior to loading, neat quercetin shows multiple sharp peaks in the XRPD curve due to its native crystalline state. No sharp peaks are seen in empty casein-chitosan complexes (S4) due to their amorphous nature. A significant change in the diffraction pattern is seen after quercetin incorporation into the polyelectrolyte complexes.