Figure 2

bLf-Dox conjugates promote greater retention of Dox resulting in enhanced cancer cytotoxicity.

(A) Representative confocal microscopy images of DU145 cells showing the time dependent internalisation of Apo-bLf-Dox and Fe-bLf-Dox studied by immunofluorescence using goat anti-bLf primary antibody (1:100) and anti-goat IgG FITC conjugated secondary antibody (1:100). The internalisation of Dox was studied using its auto-fluorescence with excitation at 488 nm and emission at 630 nm. The nucleus was counterstained with DAPI indicated by the blue fluorescence along with a competitive binding to Dox. Scale = 25 μm. (B) The percentage of cells showing green and red fluorescence co-localised in cytoplasm (yellow) was considered as percentage bLf conjugated Dox plotted as histogram mean ± S.D. (C) Histogram showing presence of nuclear red fluorescence within the cells among 100 counted cells (mean ± S.D). (D) The amount of Dox present within the cells at various time points was considered as Dox retention and the relative fluorescence was plotted against time. Measurements were performed thrice in triplicates and the results are represented as mean ± S.D. (E) The amount of Dox present in the supernatant of the cells treated at various time points was considered as Dox retention and the relative fluorescence was plotted against time. Measurements were performed thrice in triplicates and the results are represented as mean ± S.D. (F) Cytotoxicity determination based on the LDH release from the cells post 24 h treatment at different concentrations in DU145 cells. The experiment was carried out thrice in triplicates. The X-axis is represented in terms of (F). bLf protein concentrations and (G). Dox concentrations. The determination of LC₅₀ was carried out by non-linear regression fitted curve of the cytotoxicity against concentration and values are represented in the graph.