Combining CREKA-NWs with nanoworms coated with another tumor-homing peptide enhances homing efficiency. (A) Iron oxide nanoworms coated with FAM-labeled CREKA peptide were injected intravenously (5 mg of iron per kilogram of body weight) into nude mice bearing orthotropic 22Rv1 human prostate tumors. The mice had been preinjected with Ni-liposomes to reduce uptake by the reticuloendothelial system.22 Tumors were harvested 5 hours later, and tumor sections were stained with antibodies and examined by confocal microscopy (the 5-hour time point was found to be optimal for nanoworm homing with regard to accumulation of the nanoworms in the tumor and clearance of nanoworms from the blood). The CREKA-coated particles are green; blood vessels and clotting were visualized separately with anti-CD31 (magenta) or antifibrin(ogen) staining (red); nuclei were stained with DAPI (blue). Bars represent 200 μm. (B) Nanoworms coated with FAM-labeled CRKDKC or CGKRK were injected intravenously, and the tissues were collected and processed as in panel A. CRKDKC- or CGKRK-coated particles are green; blood vessels visualized with anti-CD31 are magenta (white indicates colocalization of magenta and green) and those visualized with anti-fibrin(ogen) staining are red (yellow indicates colocalization of red and green); nuclei were stained with DAPI (blue). Large vessels were selected for the panels on the right because intravascular clotting (which is not promoted by CRKDKC-NWs or CGKRK-NWs) is most apparent in larger vessels. Bars represent 200 μm (left and middle panels) and 100 μm (right panels). (C) A mixture of nanoworms coated with rhodamine-labeled CREKA (red) and FAM-labeled CRKDKC (green) was injected intravenously (2.5 mg of iron per kilogram of each nanoworm preparation), and the tissues were collected and processed as in A and stained for fibrin(ogen) (magenta); nuclei were stained with DAPI (blue). Bars represent 200 μm. (D) Mice were injected with the indicated materials as in panels A, B, or C. The sections stained with anti-fibrin(ogen) antibody were subjected to image analysis with Scanscope to quantify fibrin(ogen)-positive areas. The insets show examples of anti-fibrin(ogen) immunostaining in the tumor rim (left) and interior (right) from mice injected with the nanoworm mixture. Bars represent 50 μm. Statistical analyses were performed with analysis of variance. Error bars represent SEM (n = 5-6); **P < .01.
