Figure 4

Fluorescent images of HCT116 cells treated with free DOX, CNT-DOX-Fe₃O₄ and CNT-DOX-Fe₃O₄-Tf. (A) At 4 h exposure and at pH 7.4, DOX released from CNT-DOX-Fe₃O₄ and CNT-DOX-Fe₃O₄-Tf was observed to be localized in the nuclear region (A,B). The intracellular release of DOX can be attributed to the opening of pH-sensitive nanogates due to amide bond cleavage in the acidic lysosomal compartments. Cells incubated with free DOX showed efflux of DOX from the nucleus back into the cytoplasm, which is in contrast to the findings for CNT-DOX-Fe₃O₄ and CNT-DOX-Fe₃O₄-Tf. (B) At 4 h exposure and at pH 6.5, the fluorescence intensity of DOX from CNT-DOX-Fe₃O₄-Tf nanobot was higher due to faster cellular internalization of CNT-DOX-Fe₃O₄-Tf. (C) At 24 h and at pH 7.4, most of the DOX was released from CNT-DOX-Fe₃O₄-Tf suggesting the efficient release of DOX from interior cavity of CNT after opening of Fe₃O₄ nanogate in lysosomal conditions. (D) At 24 h and at pH 6.5, the fluorescence intensity of DOX in the cells was more pronounced suggesting enhanced cellular internalization of CNT-DOX-Fe₃O₄-Tf nanobot (Scale bars indicate 20 μm). (E) Kinetic study of Fe₃O₄ NP uncapping and DOX release from CNT-DOX-Fe₃O₄-Cy5-Tf nanobots in cells using confocal microscopy. Time-dependant release of DOX (red) into the acidic lysosomal compartment (green, LysoTracker) over 4 h, indicating -cleavage of CNT- Fe₃O₄ amide-bond, subsequent uncapping and DOX release. The merged image of the cells at 4 h shows a prominent yellow-orange signal indicating co-localization of DOX and lysosomes around the nucleus (blue), scale bars indicate 10 µm.