*2.6. In Vitro Biological Evaluation of Free and Doxorubicin-Loaded Sericin Nanocarriers* 2.6.1. Cell Culture Model

MCF-7 human epithelial mammary gland cell line (ATCC® HTB-22™) was employed in this study as in vitro model since it retains several characteristics of differentiated mammary epithelium including the ability to process estradiol via cytoplasmic estrogen receptors and the capability of forming domes. Moreover, the MCF-7 cell line is positive for the estrogen and the progesterone receptor and negative for the HER2 marker. The MCF-7 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic–antimycotic solution (ABAM, containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg amphotericin B) and maintained at 37 ◦C in a humidified air atmosphere of 5% CO2 throughout the experiment. The medium renewal was carried out every other day.

#### 2.6.2. Cell Viability Assay

The MTT assay was used to measure MCF-7 breast cancer cells' viability after incubation with Ser NPs and Ser NPs + DOX at a final concentration of 20 mg/mL Ser NPs ± Dox. Briefly, MCF-7 cells were seeded at an initial density of 2 × 105 cells/cm2 in 96–well culture plates and treated with 20 mg/mL Ser NPs and Ser NPs + DOX for 6 and 24 h. At each time point, the culture media was discarded and replaced with a freshly prepared solution of MTT (1 mg/mL). The samples were further incubated for 4 h in standard cell culture conditions to allow the metabolically active cells to form formazan crystals, which were dissolved in DMSO. The absorbance of the resulting solutions was measured at 550 nm using a Flex Station III multimodal reader (Molecular Devices).

#### 2.6.3. Cytoskeleton Investigation and DAPI staining

To evaluate the potential morphological modifications induced in the MCF-7 breast cancer cells by the treatment with Ser NPs + DOX, the cytoskeleton's actin filaments were stained with FITC–phalloidin. In this view, MCF-7 cells were seeded at an initial cell density of 2 × <sup>10</sup><sup>5</sup> cells/cm2 in 96–well culture plates and treated with 20 mg/mL Ser NPs and Ser NPs + DOX for 6 and 24 h. At each time point, the MCF-7 monolayers were fixed with a 4% paraformaldehyde solution for 20 min, permeabilized with a 2% BSA/0.1% Triton X100 solution for 1 h and consequently stained with FITC–conjugated phalloidin for 1 h at 37 ◦C in a humidified environment. In the end, the MCF-7 monolayers were stained with DAPI to highlight cell nuclei and reveal chromatin fragmentation. The samples were

imaged using the Olympus IX73 inverted fluorescence microscope (Olympus) and images were captured using CellSense Imaging Software.

### 2.6.4. Measurement of DNA Damage by Comet Assay

The DNA damage induced by the Ser NPs + DOX in MCF-7 breast cancer cells was quantified at a single cell level using OxiSelect Comet Assay Kit three-well slides (Cell Biolabs) assay. The MCF-7 cells were seeded in six-well plates at an initial density of 0.3 × <sup>10</sup><sup>6</sup> cells/cm<sup>2</sup> and treated with simple Ser NPs and DOX-loaded Ser NPs. After 6 h and 24 h, the cells were detached from the culture vessels and processed as recommended by the manufacturers' instructions. Briefly, cells were resuspended in agarose in a 1:10 ratio and transferred on the agarose precoated comet assay slides. After gelling, the comet assay slides were immersed in the lysis buffer for 60 min at 40 ◦C in the dark, followed by alkaline solution treatment for 30 min in the same conditions. Then, the slides were placed in the electrophoresis solution and then transferred to the electrophoresis tank. The electrophoresis was carried out at 1 V/cm for 15 min. Finally, samples were stained with 100 μL Vista Green DNA Dye solution for 15 min at room temperature in the dark. In total, 150 randomly selected cells from each slide were analyzed using a fluorescence microscope (Olympus IX73) and CellSense software. The length of the comet tail was chosen as an indicator of DNA damage.
