Figure 3

Stability of DNAzyme in mammary tumors, in vitro , and in vivo. (A) Stability of DNAzyme in mammary tumors. Mammary tumors were injected (as described in Materials and methods) with fluorescently-labeled AM9D and resected at either (a) 7 days, (b) 10 days, or (c) 14 days post-injection; (d) DNAzyme injected into the 2R tumor of a mouse was found to be distributed to an adjacent, non-injected mammary tumor, 3R, which emerged after intratumoral injections were first initiated. Scale bar is equivalent to 100 µm. (B) Urea-polyacrylamide gel electrophoresis of cleaved MMP9 RNA by AM9D. AM9D was incubated in PBS at 37ºC for 14 days; an equal amount was removed at days 1, 3, 5, 7, 10, and 14 (1D to 14D, respectively) and incubated with MMP9 RNA substrate at 37ºC for 2 hours. The products were then visualized on a 4% urea-polyacrylamide gel. Lane 1, RNA substrate alone; lane 2, AM9D without prior incubation at 37°C (0) cleaved RNA substrate into two fragments. AM9D incubated at 37°C for 1, 3, 5, 7, 10, and 14 days, lanes 3 to 8 respectively, did not lose its catalytic activity toward RNA substrate. (C) Stability of AM9D in vivo. The MDA-MB-231 cells were transfected with Oregon Green fluorescently labeled AM9D for 72 hours, and fixed and analyzed for the uptake and stability of AM9D molecule in the cells by fluorescent microscopy (400× magnification). (a) The nucleus is stained with 4',6-diamidino-2-phenylindole (DAPI) and (b) AM9D is shown in green. (c) The overlap of AM9D with DAPI staining indicates that AM9D is present in both the cell cytosol and nuclei, as shown by the arrow.