## 2 Materials and Methods
### Characterization of Hybrid Materials
#### 2.2.3 Encapsulation Efficiency and Drug Release Study  
The encapsulation efficiency loading of Si-Q5 and SiQ5-HNO\({}_{3}\) was evaluated by calculating the mass ratio of the total amount of encapsulated drug to the initial drug amount added to Si and Si-HNO\({}_{3}\) matrices. To calculate the total amount of the encapsulated drug, 5 and 10 mg of SiQ5 and SiQ5-HNO\({}_{3}\), respectively, were mixed with 5 mL of ethanol and underwent ultrasound-accelerated maceration for 30 min using an ultrasound bath Elmasonic S 15 H (Elma Schmidbauer GmbH, Singen, Germany) and centrifuged for 10 min at 3000\(\times\) g [32]. The supernatants were analyzed using a T65 UV-visible Spectrophotometer PG instruments (Leicestershire, UK) at 253 nm for SiQ5 and 299 nm for SiQ5-HNO\({}_{3}\). A cali  
Figure 2: Flowchart depicting the synthesis of SiQ5 with and without HNO\({}_{3}\).  
bration curve was built for quercetin extracted using SiQ5 and SiQ5-HNO3 from 5.0 mg/mL to 35 mg/mL. The encapsulation efficiency (EE%) was calculated using the formula:  
\[\mathbf{EE(\%)}=\frac{\mathbf{Wt}}{\mathbf{Wi}}\;\times\;100\]  
where Wt is the total mass of the incorporated quercetin and Wi is the total quercetin mass added initially during the sol-gel synthesis [33].  
Successively, kinetic measurements of quercetin drug release from SiQ5 and SiQ5-HNO3 were carried out in SBF at pH 7.4 and 5.0 at 37 \({}^{\circ}\)C using a T65 UV-Visible Spectrophotometer with a wavelength range of 190-1100 nm and an 8-cell motorized cell changer, purchased from PG instruments, Leicestershire (UK). The release kinetics were studied in simulated body fluid (SBF) at pH 7.4 to mimic the physiological conditions of a healthy cellular environment, and at pH 5.0 to simulate the conditions found in tumor cells [34]. For the SiQ5 system synthesized without a catalyst, a calibration curve of standard quercetin was constructed based on its \(\lambda\) max at 253 nm at pH 7.4 and 5.0, yielding an R\({}^{2}\) value of 0.9975 and 0.9995, respectively. For the silica system synthesized in the presence of nitric acid, previous studies demonstrated that quercetin undergoes oxidation during the encapsulation process, and its oxidized form was utilized to monitor the release in the presence of acid. A calibration curve of oxidized quercetin was established by measuring the absorbance of a standard concentration of oxidated quercetin prepared in SBF at pH 7.4 and 5.0 and correlating the solution concentrations with absorbance at a wavelength of 299.0 nm, resulting in an R\({}^{2}\) value of 0.9978 and 0.9994, respectively (Figure 3). The concentration range covered was from 5.0 mg/L to 35.0 mg/L. For the release test, 15 mg of both SiQ5 and SiQ5-HNO3 were solubilized in 25 mL of SBF solution at pH = 7.4 and pH 5.0 alternately while being continuously stirred and then centrifuged. The supernatant was collected for quantification via UV-Vis. The percentage quercetin release was calculated with respect to the incorporated amount of drug determined by EE for the SiQ5 and SiQ5-HNO3. The absorbance of quercetin was measured in accordance with each calibration curve in triplicate at time intervals of 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 24 h, and the outcomes were extrapolated using the calibration curve.  
Figure 3: Calibration curve UV absorbance of quercetin and oxidated quercetin at pH 7.4 and 5.0.