Chitosan-Based Nanoparticles for Intracellular Delivery of ISAV Fusion Protein cDNA into Melanoma Cells: A Path to Develop Oncolytic Anticancer Therapies

Figure 2

NP-ISAV characterization and effects. (a) Complex ensemble was evaluated between chitosan and pIRES-ISAV vector by electrophoretic migration on an agarose gel. DNA ladder (L) is shown in lane 1, naked vector in lane 2, and complexes in lane 3. Representative histograms from physical parameters such as (b) size and (c) zeta potential are shown. (d) The expression of ISAV fusion protein (upper panel) and housekeeping GAPDH (lower panel) was determined by RT-PCR 48 hours posttransfection. A representative gel shows DNA ladder (L, lane 1), PCR blank control (Blank, lane 2), parental cells nontransfected (n/transf, lane 3), Lipofectamine-ISAV cells transfected (Lipo-ISAV, lane 4), and NP-ISAV cells transfected (NP-ISAV, lane 4). (e) The fusogenic activity of the ISAV-F protein was determined by evaluating the presence of syncytia 48 h posttransfection comparing B16 parental cells (upper panel), Lipo-ISAV transfected B16 cells (middle panel), and NP-ISAV transfected B16 cells (lower panel). Cells were stained with DAPI (left column) and CellMask (middle column). The merge of both colors is shown in the right column, where a white arrow indicates a syncytium. (f) Numbers of syncytia should be quantified in B16, B16 Lipo-ISAV, and B16 NP-ISAV and correspond to the average of 5 fields, of 3 independent experiments. (g) The effect of transfection on cell viability was evaluated at 24, 48, and 120 h posttransfection and was normalized against nontransfected cells; data from Lipofectamine (Lipo-ISAV) and nanoparticle (NP-ISAV) transfected cells were graphed as . Statistical analyses were performed using the Mann–Whitney test ().

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