*2.7. In Vitro Cell Culture Studies*

In vitro cell culture studies were performed on the human breast cancer cell line (MDA-MB-231) obtained from ATCC (HTB-26™) and cultured at CERRMA (Center of Excellence for Research in Regenerative Medicine and its Applications), Faculty of Medicine, Alexandria University. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) high glucose, enriched with (10 % *v*/*v*) fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin). Cells were maintained in a 5% CO2 incubator at 37 ◦C.

### 2.7.1. MTT Cytotoxicity Assay

MDA-MB-231 cells were grown as monolayer cultures in the culture media mentioned above and monitored daily using the phase-contrast inverted microscope (CKX41SF; Olympus) for their growth and morphology. Media were changed every 2–3 days. When confluent, cells were plated into 96-well plates with a uniform seeding density of <sup>5</sup> × <sup>10</sup><sup>3</sup> cells/well and allowed to adhere for 24 h. The medium was then replaced with medium containing different concentrations of FS, FS-NS, and LF-FS-NS ranging from 10 to 100 μg/mL and the similarly diluted blank formulations NS and LF-NS, and then incubated for 48 h at 37 ◦C and 5% CO2. The media were then aspirated, replaced by MTT solution, and incubated

for another 4 h. Finally, the formazan blue crystals were dissolved in DMSO, and the absorbance was measured at 570 nm by an ELISA well-plate reader (Tecan, Infinite F50, Männedorf, Switzerland). The values obtained were compared with the control, which was regarded as 100% living cells. Experiments were performed in triplicate [26].

### 2.7.2. Apoptosis Assay (Annexin V-FTIC/Propidium Iodide Assay)

Annexin V assay was used to assess the apoptotic effect of FS, FS-NS, and LF-FS-NS [26]. Cells were incubated in 6-well plates in a density of 2 × 105 cells/well and left to adhere for 24 h at 37 ◦C and 5% CO2. Cells were treated with samples equivalent to half the IC50 value calculated from the cytotoxicity experiment for 24 h. Cells were then trypsinized, collected by centrifugation at 2000 rpm, and stained with annexin V-fluorescein isothiocyanate (FITC) (50 μg/mL, 5 μL) and propidium iodide (PI) (50 μg/mL, 5 μL) as per the manufacturer's protocol. Analysis of apoptotic cells was performed by 20,000 cells gating by flow cytometry (BD FACSCalibur™ flow cytometer, San Jose, CA, USA). The experiment was performed in triplicate with representative images provided.

### 2.7.3. Cell Migration by Wound Healing Assay

The effect of FS, FS-NS, and LF-FS-NS on the migration ability of MDA-MB-231 cells was examined by the wound healing assay [37]. Cells were seeded in 12-well plates and incubated at 37 ◦C with 5% CO2 until confluent. A 100 μL pipette tip was used to make a single scratch in the cell monolayer, and an image of the scratch was captured using the phase-contrast inverted microscope (CKX41SF; Olympus). The cells were then treated with concentrations corresponding to half IC50 values and incubated for 24 h. Following incubation, the scratch width was imaged. Images were analyzed using NIH ImageJ software, and the ratio of cell migration was calculated as the percentage of the remaining cell-free area in comparison to the area of the initial scratch.

### 2.7.4. Cellular Uptake

Fluorescently labeled NS and LF-NS were prepared using coumarin 6 (C6). A similar loading procedure was adopted with the addition of the dye to ethanol instead of FS. Briefly, 100 μL of freshly prepared C6 solution (250 μg/mL in ethanol) was added to 2 mL of NS and stirred overnight. MDA-MB-231 cells were seeded in 6-well plates at an initial density of 8 × <sup>10</sup><sup>4</sup> cells/well on coverslips. Then, 24 h later, C6-labeled NS formulations, both uncoated and LF-coated, and the free dye were added to the corresponding wells and incubated at 37 ◦C and 5% CO2 for 4 h. The cells were then washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 30 min in the dark. Cell nuclei were stained with Hoechst 33,342 for another 20 min. The cells were observed using laser scanning confocal microscopy (LEICA, DMi8, Mannheim/Wetzlar, Germany), and the fluorescent signals of the conjugated formulations were analyzed and compared with the control and free C6. Image processing was conducted using the Leica Application Suite X (LAS X) software. The uptake study was conducted in triplicates. Finally, quantification of fluorescence intensity in the obtained images was performed using ImageJ software, National Institute of Health, NIH (version 1.45s) [38].
