Fig 1.
Chemical structure of iRGD-MMAF conjugates.
(A) iRGD-MMAF. (B) FAM-iRGD-MMAF.
Fig 2.
αv integrin-dependent internalization and cytotoxicity of iRGD-MMAF.
(A) Confocal images of αv integrin-positive M21L4 and αv integrin-negative M21L human melanoma cells treated with FAM-labeled iRGD-MMAF (green). Scale bars, 50 μm. Blue, DAPI. Representative images from 3 biological replicates are shown. (B) Confocal images of KRAS-Ink mouse PDAC cells treated with FAM-labeled free MMAF or iRGD-MMAF in the presence or absence of an anti-αv integrin blocking Ab or a control IgG. Green, FAM-labeled drugs; red, cell membrane; blue, DAPI. Scale bars, 20 μm. Representative images from 3 biological replicates are shown. (C) Cytotoxicity of plain MMAF and iRGD-MMAF at 10 and 25 μM was assessed in AA0779−1 mouse CAFs and KRAS-Ink cells at different time points. (D) αv integrin-dependent cytotoxicity of MMAF or iRGD-MMAF. AA0779−1 cells were treated for 1.5 hr with 10 or 25 μM of MMAF or iRGD-MMAF in the presence or absence of an anti-αv integrin blocking Ab or a control IgG. Results from 3 biological replicates are shown. Error bars, mean ± standard error. Statistical analysis, 2-way ANOVA; ***p < 0.0005, ****p < 0.0001.
Fig 3.
Tumor-specific homing and anti-tumor effects of iRGD-MMAF.
(A) Fluorescence images showing tumor-specific homing of FAM-iRGD-MMAF. Mice bearing subcutaneous KRAS-Ink tumors were intravenously injected with 4 μmol/kg FAM-MMAF or FAM-iRGD-MMAF (green). Tissues were harvested 1 hr later and imaged under a fluorescence imager. Dash lines outline the tissues. Representative images from 3 biological replicates are shown. (B) Confocal images of the PDAC tumors shown in (A). Green, FAM-labeled drugs; red, CD31; blue, DAPI. Scale bars, 50 μm. (C) Tumor treatment study with plain MMAF and iRGD-MMAF. Mice bearing subcutaneous KRAS-Ink tumors were intravenously treated with 4 μmol/kg of MMAF or iRGD-MMAF every other day for 9 days. Control and iRGD-MMAF: n = 3, MMAF: n = 4. Error bars, mean ± standard error. Statistical analysis, one-way ANOVA; *p < 0.05. (D) Body weight changes of the mice treated in (C).
Fig 4.
Tumor-specific delivery of a co-injected dye with iRGD-MMAF.
Mice bearing subcutaneous KRAS-Ink tumors were intravenously injected with 100 μl of 1% Evans blue followed 5 min later by 4 μmol/kg of MMAF or iRGD-MMAF or an equivalent volume of PBS. Tissues were harvested 40 min later after cardiac perfusion with PBS containing 1% BSA. Representative results from 3 biological replicates are shown. (A) Images of the harvested tissues. The right-most panels show cross section images of the tumors that were sliced at 1.3 mm thickness. (B) Evans blue in the tissues was extracted and quantified based on absorbance at OD600. The values were normalized to tissue wet weight. Error bars, mean ± standard error. Statistical analysis, 2-way ANOVA; **p < 0.01.