Statistical analyses were performed with Student’s 0.05; ** 0.01; *** 0.001. Open in a separate window Fig. poor penetration into tumor tissue and by their adverse effects on healthy cells, which limits the dose of drug that can be safely administered to cancer patients. In solid tumors, many anti-cancer drugs penetrate only 3-5 cell diameters from the blood vessels, leading to reduced efficacy and the development of drug resistance (1, 2). We recently identified a tumor-penetrating peptide, iRGD (CRGDK/RGPD/EC), that when chemically conjugated to a drug can carry Procaterol HCl the drug deep into extravascular tumor tissue (3). Like conventional RGD peptides, iRGD homes to tumors by initially binding to v integrins that are specifically expressed around the endothelium of tumor vessels (3, 4, 5). iRGD is usually then proteolytically cleaved in the tumor to produce CRGDK/R. The truncated peptide loses much of its integrin-binding activity, but gains affinity for neuropilin-1 (NRP-1) because of the C-terminal exposure of a conditional C-end Rule (CendR) motif (R/KXXR/K) (6). The NRP-1 binding triggers tissue penetration, which is usually tumor specific because the cleavage requires prior binding of the peptide to integrins. These features confer on iRGD a tumor-specific tissue penetration activity. Here we have explored whether the iRGD peptide can enhance cancer drug delivery and activity when it is administered as a combination therapy with drugs that are not chemically conjugated to it. This would be advantageous because already approved drugs could be used without creating a new chemical entity, and because coupling often interferes with drug activity The activity of peptides and proteins that bind to NRP-1 through a C-terminal CendR motif can be exhibited by monitoring vascular permeability (6-8). We examined whether the CendR-triggered vascular permeability may play a role in the iRGD tumor penetrating activities using human tumor xenografts growing in immunodeficient mice and in mouse tumors growing in transgenic mice. Indeed, chemically synthesized iRGD peptide, when co-injected Procaterol HCl with the albumin-binding dye Evans Blue (9-11), caused tumor-specific accumulation of the dye in all 5 tumor models we tested (1 breast and 2 prostate cancers, and 2 pancreatic adenocarcinomas), including secondary invasion sites and disseminated tumors (fig. S1A and fig. S2, A and B). The accumulation of the dye was dependent on the dose of iRGD administered, and peaked at about 400% over the dye alone (fig. S1B). Non-CendR RGD peptides, RGD-4C (CDCRGDCFC) (12) and cyclo(-RGDfK-) (13), and a scrambled iRGD with no CendR motif (CRGDDGPKC) did not increase the permeability (fig. S1C and fig. S2C). Pre-injection of an anti-NRP-1 antibody inhibited the iRGD-induced permeability (fig. S1D). Procaterol HCl CRGDK, the truncated iRGD peptide with an uncovered CendR motif, enhanced in a dose-dependent manner local vascular permeability in the skin (fig. S3) (6). When injected systemically, CRGDK increased permeability in the tumors as well as in the lungs and heart. CRGDK was somewhat selective for the tumors, likely because of its residual affinity to integrins and the generally high expression of NRP-1 in tumors (fig. S1C) (3, 6, 14). The tumor-specific increase in tissue access mediated by iRGD suggested a way of improving the delivery of compounds to tumor parenchyma (fig. S4). A fluorescein-labeled non-CendR peptide CRGDC (FAM-CRGDC; 1.3 kDa), which minimally penetrates tumors by itself (3, 15), showed enhanced extravascular distribution upon iRGD co-injection (fig. S5A). The co-injection caused a 300% increase in both the FAM-CRGDC-positive areas (fig. S5B) and spreading of FAM-CRGDC (fig. S5C) in the tumors. Comparable results were obtained with 3 kDa and 10 kDa dextrans (fig. S5). Two nanoparticles, iron-oxide nanoworms (16) and T7 phage, also extravasated and showed enhanced accumulation in the tumor upon iRGD co-injection (fig. S6). These results show that iRGD can increase the tumor accumulation of compounds with different sizes and chemical properties. Intravenously injected molecules and nanoparticles KIAA0564 slowly extravasate into perivascular areas in.