3.2.3 In vitro liberation and inhibition assays
The three selected antibodies were analyzed for liberation from encapsulation, and the results are shown for the two proposed formulations in figure 4.
It is possible to observe that at least ~20% of the antibodies in both formulations were liberated from the micelles, as they were detected by the indirect ELISA assay. In general, formulation F2, the more hydrophobic formulation, showed the capacity to interact with the antibodies in a stronger way than F1, explaining the release profile observed. After 24 hours, the concentrations of the antibodies were ~1.10 nM for LUP-37A10 in both formulations, ~0.70 and ~1.12 nM for LUP-37C11 in formulations F1 and F2, respectively, and ~0.91 and 1.30 for LUP-37D11 in formulations F1 and F2, respectively.
We assessed the residual inhibitory activity of the antibodies released from the micelles after 6, 8 and 24 hours. The results are shown in figure 5. For LUP-37A10, 1.10 nM of antibody was able to inhibit approximately 90% of the recombinant KLK7 activity in formulation F1, while in formulation F2, the antibody inhibited ~60% of the enzyme activity. For LUP-37C11, in formulation F1, 0.7 nM of antibody inhibited more than 95% of the KLK7 activity. In formulation F2, 1.12 nM of this antibody inhibited ~80% of the KLK7 activity. For the LUP-37D11 formulation, F1 might have changed the antibody’s kinetics; that is, ~1.10 nM inhibited no less than 20% of the KLK7 activity, while in F2, ~1.30 nM inhibited more than 85% of the enzyme activity.