Molecular Imaging Of Angiogenesis in Vx-2 Rabbit Tumors With a Novel v 3-Targeted Paramagnetic Nanoparticle at 1.5 T (2003)

Methods Nanoparticle Preparation: The αvβ3-targeted paramagnetic nanoparticle contrast agent was produced by incorporating a paramagnetic lipophilic chelate into the outer lipid membrane of a perflurocarbon nanoparticle (2) covalently-complexed to an αvβ3-integrin ligand. Nontargeted paramagnetic nanoparticles were produced in an identical fashion, but without the αvβ3-integrin ligand. In addition, αvβ3-targeted non-paramagnetic nanoparticles were produced. MRI Protocol: Male New Zealand White rabbits were implanted with a 2 by 2 mm Vx-2 carcinoma tumor fragment in the popliteal fossa. Twelve days after implantation, animals were randomized into three groups receiving either: 1) αvβ3-integrin-targeted paramagnetic nanoparticles (targeted, n=4), 2) nontargeted paramagnetic nanoparticles (control, n=4), or 3) αvβ3-integrin targeted non-paramagnetic nanoparticles followed by αvβ3-integrin-targeted paramagnetic nanoparticles (competition, n=4). All nanoparticle doses were 0.5 ml/kg and were administered via an ear vein catheter. Animals were imaged with a clinical 1.5 T scanner (NT Intera with Master Gradients, Philips Medical Systems, Best, Netherlands) using a standard 11 cm diameter circular surface coil placed against the tumor-bearing hindlimb. High-resolution, T1-weighted, fat suppressed, three-dimensional, gradient echo images (TR: 40 ms, TE: 5.6 ms, FOV: 64 mm, slice thickness: 0.5 mm, contiguous slices: 30, in-plane resolution: 250µ m, signal averages: 2, flip angle: 65 o , scan time: 15 min) were collected before and immediately, 60 and 120 minutes after paramagnetic nanoparticle injection. After MR imaging, all tumors were resected and frozen for histology to corroborate contrast results. Image Analysis: To determine changes in pixel signal intensity with time, T1-weighted images (three contiguous slices through the center of each tumor) were analyzed with MATLAB (The MathWorks, Inc, Natick, MA). Serial images were spatially co-registered and contrast enhancement was calculated for each pixel at each post-injection timepoint. An ROI was manually drawn around a portion of the hindlimb muscle in the baseline images and the average pixel-by-pixel signal enhancement inside the ROI was calculated at each timepoint. A second ROI was manually drawn around the tumor and the standard deviation of the tumor signal was calculated in the baseline image for each animal. Pixels were selected provided that the signal intensity increased by greater than three times the standard deviation of the tumor signal at baseline. These enhancing pixels were mapped back to each timepoint and the average signal increase was determined. Results T1-weighted MR images of the Vx-2 tumor rabbits receiving αvβ3-targeted paramagnetic nanoparticles revealed a marked increase in MR contrast primarily, although not exclusively, located asymmetrically along the tumor periphery (Fig. 1). αvβ3-integrin contrast enhancement was typically seen in a patchy distribution within the walls of proximate vessels and tissue fascial interfaces adjoining the tumor. Histology and immunocytochemical assessments of the Vx-2 tumors corroborated that angiogenesis was most intensely distributed within a few independent regions along the tumor periphery and found less extensively within intratumoral connective tissue tracts interspersed between tumor cell lobules. At the two hour timepoint, the magnitude of signal enhancement among rabbits treated with the αvβ3-targeted nanoparticles increased 55% relative to nontargeted nanoparticles (Fig. 2). Competitive blockage of αvβ3-integrin sites with αvβ3-targeted non-paramagnetic nanoparticles two hours before injection of the αvβ3-targeted paramagnetic particles greatly diminished contrast signal enhancement (p<0.05) at 2 hrs., confirming the specificity of the targeted nanoparticles. All animal groups displayed minimal MRI signal enhancement in the muscle (Targeted: 6.5 ± 3%, Control: 6.2 ± 3%, Competition: 8.8 ± 1.3%). Discussion In the present study, New Zealand White rabbits with nascent Vx-2 tumors were treated systemically with an αvβ3-integrin targeted paramagnetic nanoparticle agent that specifically bound and significantly increased the MR detection of the developing neovasculature using a commercially-available, clinical magnetic resonance scanner (1.5 T). Targeted contrast signal enhancement was specific to sites of angiogenesis associated directly with the tumor and adjoining vasculature stimulated by the developing cancer. Signal intensity of the targeted angiogenic endothelium increased steadily and rapidly to greater than 125% after two hours. These data suggest that MR imaging at later timepoints may yield further increased signal enhancement. Signal enhancement produced in the tumor periphery with nontargeted nanoparticles was attributed to nonspecific leakage and early retention of the contrast agent within the neovasculature. The marked detectability of the nanoparticles reflects their “ultraparamagnetic” nature with a molecular relaxivity of greater than 1,010,000