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).