2.3.3. Cell Death Pattern through Acridine Orange/Ethidium Bromide (AO/EB) Test

Considering the results observed for NCUR/MTX-2 in reducing cell viability of Calu-3, the cell death pattern of this formulation was investigated after the 24 h treatment by AO/EB staining to verify the death mechanism.

The cytotoxic effect of NCUR/MTX-2 formulations can be observed in Figure 6. The control showed viable cells with normal and bright green nuclei (Figure 6A, white arrows). NCUR/MTX-2 resulted in early-stage apoptotic cells that were marked by crescent-shaped or granular yellow-green acridine orange nuclear staining (Figure 6B, yellow arrows), and late-stage apoptotic cells showing concentrated and asymmetrically located orange nuclear ethidium bromide staining (Figure 6B, blue arrow). In addition, the occurrence of apoptotic membrane blebbing (Figure 6B, red arrow) was verified since membrane blebbing is required for redistribution of fragmented DNA from the nuclear region into membrane blebs and apoptotic bodies [53]. No necrotic cell was observed after treating the Calu-3 cells with the co-loaded formulation.

**Figure 6.** Effect of NCUR/MTX-2 on the morphology of Calu-3 cells stained with acridine orange/ ethidium bromide by light fluorescence microscopy at 400× magnification. The negative control (**A**, vehicle) presented viable cells with normal nucleus staining represented by the bright green chromatin (white arrows); NCUR/MTX-2 (**B**) showed early-stage apoptotic cells that were marked by crescent-shaped or granular yellow-green acridine orange nuclear staining (yellow arrows), late-stage apoptotic cells showing concentrated and asymmetrically localized orange nuclear ethidium bromide staining (blue arrow), and apoptotic membrane blebbing (red arrow). Images are representative of results obtained from three independent biological replicates (*n* = 6 slides per sample).

Apoptosis is typically represented by a series of intracellular events, which ultimately lead to DNA fragmentation and the internucleosomal degradation of genomic DNA due to the activation of endogenous endonucleases. This mechanism is less aggressive because apoptosis is controlled and energy-dependent and can affect individual cells or clusters of cells [54]. On the other side, necrotic cells swell and rupture, releasing the cytoplasmic material. This process is characterized by injury to a group of cells rather than by individual death. Necrosis occurs by disruption of plasma membranes and organelles, marked dilation of mitochondria with the emergence of large amorphous densities, probably representing denatured proteins. The nucleus may shrink, suffer fragmentation or even totally disappear by unspecific DNA fragmentation [55]. In that sense, the co-loaded formulation was able to provide a Calu-3 death pattern by a carefully regulated energy-dependent process, characterized by programmed cell death, which is a widespread component of both health and disease. Moreover, apoptosis is a superior mechanism for feasible therapeutic interventions on the pathophysiology of lung cancer.
