## 2 Materials and Methods
### In Vitro Cytotoxicity Analysis  
#### 2.10.1 Cell Viability  
Cell proliferation was analyzed through MTT assays against the MG-63 human osteosarcoma cell line. The cell line was procured from NCCS, Pune, India. The cells were maintained in DMEM high glucose media (AL111, HIMEDIA) supplemented with 10% FBS (RM10432, HIMEDIA) along with a 1% antibiotic-antimycotic solution in an atmosphere of 5% CO\({}_{2}\), 18-20% O\({}_{2}\) at 37 \({}^{\circ}\)C temperature in a CO\({}_{2}\) incubator and sub-cultured every 2 days. A 100 \(\mu\)L cell suspension was seeded in a 12-well plate with a cell density of 50,000 cells per well without the test agent and the cells were allowed to grow for 24 h. The samples were sterilized by keeping them under UV radiation for 30 min and gently washed twice with 1\(\times\) PBS. The samples were then placed in each well of a 12-well plate with sterile forceps and the wells were filled with fresh culture medium. Cells without any sample were considered as untreated and cells treated with doxorubicin with 1 uM/mL concentration were considered as a positive control for the study. Afterward, the plates were incubated for 48 h at 37 \({}^{\circ}\)C in a 5% CO\({}_{2}\) atmosphere. After the incubation period, the plates were taken out from the incubator and the spent media was removed. MTT reagent (HIMEDIA) was added to a concentration of 0.5 mg/mL of the total volume. The plates were returned to the incubator and incubated for 3 h. After incubation, the MTT reagent was removed and a solubilization solution (DMSO) was added followed by gentle stirring in a gyratory shaker to enhance dissolution. The absorbance was calculated in an ELISA plate reader (Thermo Fisher) at 570 nm and the final cell viability was calculated using Equation (4).  
\[\%\;\text{Cell Viability}\;\;\;\frac{\textit{Mean abs. of treated cells}}{\textit{Mean abs. of untreated cells}}\times 100 \tag{4}\]  
The viability of the cells on the developed samples was confirmed through confocal microscopic analysis ([PERSON], Oberkochen, Germany). Cells were cultured in a 12-well plate at a density of 2 \(\times\) 10\({}^{5}\) cells/2 mL and incubated in a CO\({}_{2}\) incubator at 37 \({}^{\circ}\)C for 24 h. The samples were sterilized under UV radiation and then placed in each well with the help of sterile forceps. At the end of the treatment, the medium was removed from the wells and they were washed with PBS. Afterward, the cells were trypsinized with 300 \(\mu\)L trypsin-EDTA solution and were harvested into 2 mL Eppendorf tubes. The tubes were centrifuged at 2000 rpm for 5 min. The cells were stained with 200 \(\mu\)L of staining solution for 10 min. The staining solution was then removed by centrifuging the tubes at 1000 rpm for 5 min and washing with PBS to remove excess dye. Then, 100 \(\mu\)L of cell suspension was carefully loaded on a clean glass slide under a coverslip, and a drop of mounting medium was added before imaging. Observations were made under a fluorescence microscope with a filter cube with excitation 560/40 nm and emission 645/75 nm for EtBr and excitation 470/40 and emission 525/50 for acridine orange. Finally, the images were overlayed by ImageJ Software v1.48.