Vascular disruption in the mouse Matrigel model with αvβ3-targeted RGD-Dox-NP. Mice were injected s.c. on the flank with Matrigel containing 400 ng of human recombinant bFGF. NPs containing 1 mg/kg of Dox were i.v.-injected on days 1, 3, and 5. (A) After the treatment, mice were i.v.-injected with fluorescein-labeled G. simplicifolia lectin and the plugs were removed and imaged by scanning confocal microscopy. (B and C) To quantify angiogenesis, the matrigel plugs were removed and the fluorescent-lectin content was quantified with a fluorimeter. *, P < 0.05 for RGD-Dox-NP vs. PBS. (Scale bar: 100 μm.)

In vitro and in vivo αvβ3 targeting of RGD-NP. (A) Schematic representation and transmission EM of Dox-loaded RGD-NP. (B) Competition assay for in vitro αvβ3 targeting of RGD-NP in endothelial cells. HUVECs were pretreated for 5 min with a 20-fold molar excess of either cRGDfK or cRADfK to test for inhibition of NP binding. Subsequently, the cells were incubated with the RGD-NPs for 20 min, and binding was studied by scanning confocal microscopy for the BODIPY 630/650 dye. (C) In vivo integrin αvβ3 targeting of RGD-NPs within the tumor neovasculature was studied by intravital microscopy with the dorsal skin-fold window chamber. M21L melanomas (αvβ3 negative) were allowed to vascularize for 7 days, and mice were i.v.-injected with 200 nmol of either RGD-NP or RAD-NP containing BODIPY 630/650. NPs were imaged by confocal scanning microscopy at 5-h postinjection. GFP-labeled M21L melanomas are shown in green, and NPs are in blue. (Scale bars: A, 100 nm; B and C, 100 μm.)

RGD-Dox-NP reduces overall metastasis in an orthotopic renal cell carcinoma model. (A) Human SN12C-RFP cells were injected into the left kidney and primary tumors were established for 8 days. At that time, NPs containing 2 mg/kg Dox were dosed every other day from day 8 until day 42. At the endpoint, both kidneys were removed and the difference in weight was attributed to primary tumor burden. Additionally, the diaphragm, liver, pancreas, and spleen were resected and imaged for RFP-expressing metastatic lesions by using an OV-100 whole animal imaging system. The total pixels in each image were quantified by using ImageJ software and the average pixels of the metastatic lesions/animal are represented as the metastatic burden. *, P < 0.05 for RGD-Dox-NPs vs. RAD-Dox-NPs. (B) Images of the RFP fluorescence from the metastatic lesions present in the diaphragm, liver, pancreas, and spleen used for the quantification of the metastatic burden in A are presented for each animal. (Scale bars: 5 mm.)

Suppression of metastasis in an orthotopic model of pancreatic cancer. (A) R40P pancreatic carcinoma cells were injected into the tail of the pancreas in Tie-2 GFP mice and allowed to grow for 11 days. RGD-NPs labeled with BODIPY 630/650 were i.v.-injected, and the primary tumor, pancreas, liver, lung, kidney, and heart were imaged by confocal microscopy. Red represents rhodamine-labeled G. simplicifolia lectin for staining the endothelium, and green represents NP binding. (B) Quantitation of the fluorescent NPs in various tissues expressed as the percent of pixels from the confocal images. (C) Immunohistochemistry of serial sections from the tumors treated with the RGD-Dox-NPs demonstrates areas of apoptosis (TUNEL staining) that colocalize only with blood vessels that express the target (β3). (D) Integrin αvβ3-targeted RGD-Dox-NPs prevent metastasis to the hepatic hilar lymph node. After surgical implantation of the cells, mice were treated on days 5, 7, and 9 with RGD-Dox-NP, RAD-Dox-NP, free Dox, or PBS (each with 1 mg/kg total Dox per dose). On day 11, the primary tumor and the hepatic hilar lymph node were resected and weighed. (E) Effect of free Dox at various concentrations (1, 7.5, and 15 mg/kg dosed on days 5, 7, and 9) on primary tumor growth and metastasis to the hepatic hilar lymph node. *, P < 0.05 for RGD-Dox-NP vs. PBS. **, P < 0.05 for 15 mg/kg free Dox vs. PBS. (Scale bars: A and C, 100 μm.)