The purpose of this study was to develop a bifunctional iron oxide (IO) nanoparticle probe for PET and MRI scans of tumor integrin a v b 3 expression. Methods: Polyaspartic acid (PASP)-coated IO (PASP-IO) nanoparticles were synthesized using a coprecipitation method, and particle size and magnetic properties were measured. A phantom study was used to assess the efficacy of PASP-IO as a T2-weighted MRI contrast agent. PASP-IO nanoparticles with surface amino groups were coupled to cyclic arginine-glycine-aspartic (RGD) peptides for integrin a v b 3 targeting and macrocyclic 1,4,7,10-tetraazacyclododecane-N,N9,N$,N9$,-tetraacetic acid (DOTA) chelators for PET after labeling with 64 Cu. IO nanoparticle conjugates were further tested in vitro and in vivo to determine receptor targeting efficacy and feasibility for dual PET/MRI. Results: PASP-IO nanoparticles made by single-step reaction have a core size of 5 nm with a hydrodynamic diameter of 45 6 10 nm. The saturation magnetization of PASP-IO nanoparticles is about 117 emu/g of iron, and the measured r 2 and r 2 * are 105.5 and 165.5 (sÁmM) 21 , respectively. A displacement competitive binding assay indicates that DOTA-IO-RGD conjugates bound specifically to integrin a v b 3 in vitro. Both small-animal PET and T2-weighted MRI show integrinspecific delivery of conjugated RGD-PASP-IO nanoparticles and prominent reticuloendothelial system uptake. Conclusion: We have successfully developed an IO-based nanoprobe for simultaneous dual PET and MRI of tumor integrin expression. The success of this bifunctional imaging approach may allow for earlier tumor detection with a high degree of accuracy and provide further insight into the molecular mechanisms of cancer.

To date, the in vivo imaging of quantum dots (QDs) has been mostly qualitative or semiquantitative. The development of a dual-function PET/near-infrared fluorescence (NIRF) probe can allow for accurate assessment of the pharmacokinetics and tumor-targeting efficacy of QDs. Methods: A QD with an aminefunctionalized surface was modified with RGD peptides and 1,4,7,10-tetraazacyclodocecane-N,N9,N$,N%-tetraacetic acid (DOTA) chelators for integrin a v b 3 -targeted PET/NIRF imaging. A cell-binding assay and fluorescence cell staining were performed with U87MG human glioblastoma cells (integrin a v b 3 -positive). PET/NIRF imaging, tissue homogenate fluorescence measurement, and immunofluorescence staining were performed with U87MG tumor-bearing mice to quantify the probe uptake in the tumor and major organs. Results: There are about 90 RGD peptides per QD particle, and DOTA-QD-RGD exhibited integrin a v b 3 -specific binding in cell cultures. The U87MG tumor uptake of 64 Cu-labeled DOTA-QD was less than 1 percentage injected dose per gram (%ID/g), significantly lower than that of 64 Culabeled DOTA-QD-RGD (2.2 6 0.3 [mean 6 SD] and 4.0 6 1.0 %ID/g at 5 and 18 h after injection, respectively; n 5 3). Taking into account all measurements, the liver-, spleen-, and kidney-tomuscle ratios for 64 Cu-labeled DOTA-QD-RGD were about 100:1, 40:1, and 1:1, respectively. On the basis of the PET results, the U87MG tumor-to-muscle ratios for DOTA-QD-RGD and DOTA-QD were about 4:1 and 1:1, respectively. Excellent linear correlation was obtained between the results measured by in vivo PET imaging and those measured by ex vivo NIRF imaging and tissue homogenate fluorescence (r 2 5 0.93). Histologic examination revealed that DOTA-QD-RGD targets primarily the tumor vasculature through an RGD-integrin a v b 3 interaction, with little extravasation. Conclusion: We quantitatively evaluated the tumor-targeting efficacy of a dual-function QD-based probe with PET and NIRF imaging. This dual-function probe has significantly reduced potential toxicity and overcomes the tissue penetration limitation of optical imaging, allowing for quantitative targeted imaging in deep tissue.

In vivo imaging of a v b 3 expression has important diagnostic and therapeutic applications. Multimeric cyclic RGD peptides are capable of improving the integrin a v b 3 -binding affinity due to the polyvalency effect. Here we report an example of 18 F-labeled tetrameric RGD peptide for PET of a v b 3 expression in both xenograft and spontaneous tumor models. Methods: The tetrameric RGD peptide EfE[c(RGDyK)] 2 g 2 was derived with amino-3,6,9-trioxaundecanoic acid (mini-PEG; PEG is poly(ethylene glycol)) linker through the glutamate a-amino group. NH 2 -mini-PEG-EfE[c(RGDyK)] 2 g 2 (PRGD4) was labeled with 18 F via the N-succinimidyl-4-18 F-fluorobenzoate ( 18 F-SFB) prosthetic group. The receptor-binding characteristics of the tetrameric RGD peptide tracer 18 F-FPRGD4 were evaluated in vitro by a cell-binding assay and in vivo by quantitative microPET imaging studies. Results: The decay-corrected radiochemical yield for 18 F-FPRGD4 was about 15%, with a total reaction time of 180 min starting from 18 F-F 2 . The PEGylation had minimal effect on integrin-binding affinity of the RGD peptide. 18 F-FPRGD4 has significantly higher tumor uptake compared with monomeric and dimeric RGD peptide tracer analogs. The receptor specificity of 18 F-FPRGD4 in vivo was confirmed by effective blocking of the uptake in both tumors and normal organs or tissues with excess c(RGDyK). Conclusion: The tetrameric RGD peptide tracer 18 F-FPRGD4 possessing high integrin-binding affinity and favorable biokinetics is a promising tracer for PET of integrin a v b 3 expression in cancer and other angiogenesis related diseases.

Integrin a v b 3 plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic arginine-glycine-aspartic (RGD) peptides can be used for noninvasive imaging of a v b 3 expression and targeted radionuclide therapy. In this study, we developed 64 Cu-labeled multimeric RGD peptides, EfE[c(RGDyK)] 2 g 2 (RGD tetramer) and E(EfE[c(RGDyK)] 2 g 2 ) 2 (RGD octamer), for PET imaging of tumor integrin a v b 3 expression. Methods: Both RGD tetramer and RGD octamer were synthesized with glutamate as the linker. After conjugation with 1,4,7,10-tetra-azacyclododecane-N, N9,N$,N$9-tetraacetic acid (DOTA), the peptides were labeled with 64 Cu for biodistribution and small-animal PET imaging studies (U87MG human glioblastoma xenograft model and c-neu oncomouse model). A cell adhesion assay, a cell-binding assay, receptor blocking experiments, and immunohistochemistry were also performed to evaluate the a v b 3 -binding affinity/specificity of the RGD peptide-based conjugates in vitro and in vivo. Results: RGD octamer had significantly higher integrin a v b 3 -binding affinity and specificity than RGD tetramer analog (inhibitory concentration of 50% was 10 nM for octamer vs. 35 nM for tetramer). 64 Cu-DOTA-RGD octamer had higher tumor uptake and longer tumor retention than 64 Cu-DOTA-RGD tetramer in both tumor models tested. The integrin a v b 3 specificity of both tracers was confirmed by successful receptor-blocking experiments. The high uptake and slow clearance of 64 Cu-DOTA-RGD octamer in the kidneys was attributed mainly to the integrin positivity of the kidneys, significantly higher integrin a v b 3 -binding affinity, and the larger molecular size of the octamer, as compared with the other RGD analogs. Conclusion: Polyvalency has a profound effect on the receptor-binding affinity and in vivo kinetics of radiolabeled RGD multimers. The information obtained here may guide the future development of RGD peptidebased imaging and internal radiotherapeutic agents targeting integrin a v b 3 .

The dimeric RGD peptide tracer (68)Ga-NOTA-RGD2 with good tumor uptake and favorable pharmacokinetics warrants further investigation for potential clinical translation to image integrin alpha(v)beta(3).