2.2.5. In Vitro Studies on HaCaT and SK-Mel-28 Cell Lines

For the in vitro studies, two human-derived cell lines (Instituto Multidisciplinario de Biología Celular-CIC-CONICET, Tolosa, Argentina) were used: HaCaT, immortalized non-tumorigenic keratinocytes, and SK-Mel-28, melanoma-derived cell line. Both have the Hh signaling pathway activated [45,46]. For all experiments, HaCaTs were maintained in RPMI 1640, whereas SK-Mel-28 were maintained in MEM supplemented with 1 mM of sodium pyruvate. Both cell lines were at 37 °C with 5% CO₂ and supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B.

Cell viability after 4 and 24 h of incubation with empty UDL, UDL-Vis, and free Vismodegib (solubilized in DMSO, with a final concentration of 1% of solvent in the well) was determined by three different methods: MTT, crystal violet (CV), and neutral red (NR).

A total of 1 × 10⁴ cells were seeded on each well of 96-well flat-bottom microplates. After 24 h, the medium was replaced with 100 µL of the different formulations diluted in cell medium with FBS. The Vismodegib concentration assessed ranged from 0.04 to 0.65 mg/mL, which correspond to concentrations of SPC from 1.6 to 25.8 mM. An untreated control with cell medium with FBS and a control with cell medium with FBS plus 1% DMSO were included. After incubation, the media were removed; cells were washed three times with phosphate-buffered saline pH 7.4 (PBS); and the MTT, CV, and NR assays were performed as described in Calienni et al., 2018 [19]. Measurements were performed with a cell imaging multi-mode reader Cytation 5 (BioTek Instruments, Winooski, VT, USA). The cell viability was calculated according to the following equation, where AbsT is the absorbance of treated cells and AbsC is the absorbance of the corresponding control (without or with 1% DMSO). Data were reported as the mean of three different experiments ± SD:

The evaluation of the apoptotic induction was carried out with the Annexin V-FITC apoptosis detection kit (BD Pharmingen™, San Diego, CA, USA). Cells were seeded in 6-well plates at a density of 3 × 105 cells/well and allowed to grow for 24 h. The medium was then replaced with 2 mL of fresh medium containing UDL-Vis or free Vismodegib, the last with 1% DMSO, in a concentration corresponding to 0.32 mg/mL of Vismodegib. Cells were incubated for 4 h, washed with PBS, and trypsinized. An untreated control and a control with 1% DMSO were included. Three independent determinations were carried out for each condition. The staining was performed according to the kit instruction. A total of 1.9 × 10⁴ cells were analyzed within 1 h by flow cytometry (Becton Dickinson FACSCalibur, Franklin Lakes, NJ, USA), with FL1 and FL3 channels. Data were processed using BD CellQuest™ Pro 6.0 software (Becton Dickinson, Franklin Lakes, NJ, USA).

Evaluation of the Uptake by Flow Cytometry

The cellular uptake of F-UDL-Vis (0.32 mg/mL of Vismodegib) was monitored in HaCaT and SK-Mel-28 by flow cytometry. A total of 1 × 10⁵ cells were seeded per well in 24-well plates and allowed to grow for 24 h. On one hand, uptake kinetics was performed in duplicate to determine the optimal incubation time to carry out the study. For this, the cells were incubated with 400 µL of F-UDL-Vis diluted in culture medium for 1, 2, and 4 h at 37 °C. After the time, the cells were washed three times with PBS, trypsinized, and centrifuged at 125× g. The pellets were resuspended with 300 µL of PBS. Then, flow cytometry was performed to quantify the uptake of the F-UDL-Vis over time. The samples were excited with a 488 nm laser and the FL1 filter (530/30 nm) was used to detect TopFluor^® cholesterol.

After determining the incubation time for both lines, cells were treated with the F-UDL-Vis diluted in culture medium at 4 and 37 °C. Cells and the nanoformulation were previously incubated at the corresponding temperature for 1 h before adding the sample. A total of 1.9 × 10⁴ cells were analyzed in duplicate by flow cytometry. Untreated controls were included in the study of kinetics and uptake at 4 and 37 °C.

As a control of the cell viability, the apoptosis detection test detailed in the section “Evaluation of cell apoptosis” was performed in parallel to untreated cells incubated under the same conditions.

Evaluation of the Uptake by Fluorescence Microscopy

Cell uptake was also monitored in both cell lines by fluorescence. Cells were grown for 24 h in 24-well plates and then incubated with 400 µL of F-UDL-Vis diluted in culture medium (0.32 mg/mL of Vismodegib) for 4 h at 4 and 37 °C. Before incubating, cells, as well as the nanoformulation, were maintained at the corresponding temperature for 1 h. Three washes with PBS were performed to eliminate the remnant formulation and cells were fixed with cold methanol for 1 min. After three washes with PBS, the nuclei of cells were stained with Fluoroshield™ with DAPI for 5 min and the samples were mounted with coverslips. Untreated controls, stained with DAPI, were included.

Microscopies were carried out using a Cytation 5 configured with DAPI, GFP, and RFP filter cubes, in combination with LED light sources (365, 465, and 523 nm, respectively), to detect DAPI, TopFluor^® cholesterol, and propidium iodide, respectively. The DAPI cube was configured with a 377/50 excitation filter and a 447/60 emission filter; the GFP cube used a 469/35 excitation and a 525/39 emission filters, and the RFP cube used a 531/40 excitation and 593/40 emission filters. Exposure settings were automatically determined for each color and were the same for all samples. The focus was set automatically using the DAPI signal as a reference.