# 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 loading of quercetin in the casein/chitosan gel particles can be proven with FT-IR spectroscopy (Figure 4). The characteristic peaks for quercetin are as follows: band in the region between 3400 cm\({}^{-1}\) and 3200 cm\({}^{-1}\) corresponding to--OH stretching, peak at 1664 cm\({}^{-1}\) showing carbonyl group stretch in the aromatic ketone, 1611 cm\({}^{-1}\) representing aromatic bending, 1214 cm\({}^{-1}\) evident for the bending of the--OH groups in the phenol, and peak at 1092 cm\({}^{-1}\) due to the aromatic bending [36]. The presence of the same peaks and broad bands of quercetin in the final/complex spectra confirms the presence of quercetin in the casein/chitosan complexes. Thus, it can be concluded that the substance is physically incorporated within the complexes without chemical interaction between the two. The lack of any noticeable changes in both the position and shape of the quercetin peaks can  
Figure 3: AFM microphotograph (**a**) and cross-section (**b**) of casein/chitosan complex (Sample S4). Figure 2: SEM microphotography of casein/chitosan complex (Sample S4). also indicate its successful physical incorporation within the casein-chitosan network without the formation of any covalent or hydrogen bonds between the network and the incorporated substance. The loading efficiency of quercetin is very high (above 95% for all investigated samples), and it reaches a maximum value for S6 (casein/chitosan charge ratio 6:1). This behaviour is typical for hydrophobic compounds in general and for quercetin in particular. The incorporation of hydrophobic compounds in polymeric carriers is driven by the hydrophobic attractions between hydrophobic segments of the different polymers, such as the hydrophobic core of the casein micelles and the hydrophobic bioactive compounds (the polyphenol rings of quercetin) [37; 38]. The amount of quercetin released in phosphate buffer at pH 5.5 and a constant temperature of 32.0 \(\pm\) 0.5 \({}^{\circ}\)C during the first 480 min (8 h) after immersion in the medium is presented in Figure 5.  
Figure 4: FT-IR spectra of neat quercetin and blank and quercetin-loaded CAS/CH gel complexes. Figure 5: Quercetin release from CAS/CH complexes. The release during the first 8 h is incomplete, varying between 1% and 2.5% depending on the complex composition. The results demonstrate no burst effect or linear release kinetics. Analogously, very slow release of quercetin from poly (lactic-co-glycolic acid) microspheres is reported by [PERSON] and [PERSON] [39], who found that only 40% of the included drug amount is released during a month. Similar achievements were reached by [PERSON] and co-authors, who studied the quercetin release from TiO\({}_{2}\) nanotubes coated with chitosan [40]. They found about 2% release in the first 8 h. Despite the slow release rate, if we assume that the studied matrix contains 50 mg of quercetin, within 8 h, a released amount will be adequate to prevent inflammation after post-operative surgery [41]. The best fitting results of the experimental data were achieved with the zero-order kinetics model [42]:  
\[Q_{t}=Q_{0}+K_{0}t \tag{4}\]  
where \(Q_{t}\) is the cumulative amount of drug released at time \(t\), \(Q_{0}\) is the initial amount of drug, \(K_{0}\) is the release kinetic constant, and \(t\) is the time at which the drug release is measured. Usually, this model is applicable in core-shell systems, where a core containing the drug is surrounded by polymeric matrices. Drug release is mediated by diffusion through the surrounding polymeric matrices. Zero-order release is maintained because a constant concentration gradient of drug is kept in the polymeric matrices as long as the reservoir contains a saturated solution and sufficient excess of solid drug [43]. The observed type of release kinetics could be associated with the casein/chitosan gel polyelectrolyte structure, which has already been discussed.