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<p>Iron oxide nanoparticles are a useful diagnostic contrast agent and have great potential for therapeutic applications. Multiple emerging diagnostic and therapeutic applications and the numerous versatile parameters of the nanoparticle platform require a robust biological model for characterization and assessment. Here we investigate the use of iron oxide nanoparticles that target tumor vasculature, via the tumstatin peptide, in a novel three-dimensional tissue culture model. The developed tissue culture model more closely mimics the <i>in vivo</i> environment with a leaky endothelium coating around a glioma tumor mass. Tumstatin-iron oxide nanoparticles showed penetration and selective targeting to endothelial cell coating on the tumor in the three-dimensional model, and had approximately 2 times greater uptake <i>in vitro</i> and 2.7 times tumor neo-vascularization inhibition. Tumstatin provides targeting and therapeutic capabilities to the iron oxide nanoparticle diagnostic contrast agent platform. And the novel endothelial cell-coated tumor model provides an <i>in vitro</i> microtissue environment to evaluate nanoparticles without moving into costly and time-consuming animal models.</p>