3.2.4 Morphology and MTT assays
After all these tests, we also decided to perform a cytotoxicity test to investigate the reliability of these antibodies for use in potentialin vivo therapy. Vero cells were used for cytotoxicity assays, because they are the standard cell used in international studies with biomaterials, and they have fibroblast-like features (Lombello, Malmonge, & Wada, 2000; Nascimento et al ., 2017). Vero cells presented a morphological pattern very similar to the growth pattern of the cells on the culture plate, forming a confluent monolayer of cells with predominantly elongated morphology, thereby indicating no cytotoxicity induced by the antibodies.(Nascimento et al., 2017) It was also observed, through the images obtained by light microscopy, that the core presented with one or more nucleoli and vacuoles and had proliferated to a high cell density.
The results of the MTT assay for cell viability confirmed the noncytotoxic characterization of the antibodies. The principle of the method was to reduce a yellow biomaterial, salt tetrazolium MTT, by mitochondrial enzymes, resulting in a blue/purple product called formazan, which may be quantified by spectrophotometry. Thus, this reaction occurs only in living cells and is activated in the mitochondria, thereby serving as a versatile and quantitative method for assessing cell viability (Mosmann, 1983; Shen et al ., 2010).
Figure 6 and 7 how the results of the cytotoxicity and morphology assays, respectively. Figure 6 shows that the antibodies did not show cytotoxic activity in the Vero cells. The cell viability remained greater than 80% for all the antibodies tested in all formulations. Figure 7 presents the morphological effects of the antibodies on the Vero cells. It was possible to observe that the cells had conserved their fibroblast-like features.
The cytotoxicity induced by the scFv-type antibodies is mostly studied in multiple types of cancer, specifically for tumor targeting (Huttet al ., 2018; Klement et al ., 2017; Marty & Schwendener, 2005; Singh et al ., 2007; Sokolowska-Wedzina et al. , 2017); in these cases, it is interesting that antibodies display certain cytotoxic effects towards the cancer cells. In our case, however, the antibodies should be capable of only inhibiting the enzyme without inducing cytotoxicity. Little is known about the effects of the scFv-Fc antibodies alone; however, Hutt et al . were able to demonstrate cytotoxic activity of scFv-Fc-scTRAIL fragments, which promoted cell apoptosis (Hutt et al ., 2018), and Sokolowska-Wedzina et al . were able to conjugate a scFv-Fc antibody with a cytotoxic drug for delivery to lung cancer cells (Sokolowska-Wedzina et al ., 2017).