The purpose of the study is to develop targeted MRI contrast agent for molecular imaging of aggressive prostate cancer (PCa). 1 in 7 men in the United State has a lifetime risk of being diagnosed with PCa. Accurate detection of PCa is critical for improving survival of patients diagnosed with high-risk PCa. Prostate-specific antigen (PSA) screening is routinely used in PCa detection, but the non-specificity of PSA screening to malignant cancer types resulted in overtreatments with serious adverse effects. Extradomain-B fibronectin (EDB-FN), an isoform of fibronectin, is substantially upregulated in the extracellular matrix (ECM) of aggressive PCa, with undetectable expression in normal tissue. EDB-FN promotes cancer invasion and migration via interacting with tumor cells and other ECM molecules. Here we set out to construct a MRI contrast agent that targets to EDB-FN with the aid of a 7-amino acid peptide, ZD2. Specific targeting to overexpressed EDB-FN can increase sensitivity for MRI detection of aggressive PCa, while minimizing false-positive detection associated with indolent disease. ZD2 was discovered with phage display technique as reported previously¹. The peptide sequence, TVRTSAD, was synthesized in solid phase using Fmoc chemistry. A short PEG spacer (Fmoc-12-amino-4,7,10-trioxadode-canoic acid), and 5-hexynoic acid, were also sequentially conjugated in solid phase followed by trifluoroacetic acid (TFA) treatment, which yielded ZD2-PEG-propargyl for click chemistry. Azido-Gd(HP-DO3A) was synthesized as reported previously². Click chemistry reaction between ZD2-PEG-propargyl (1 Eq) and azido-Gd(HP-DO3A) (1.1 Eq) was performed in a 2:1 mixture of t-butanol and water under nitrogen following addition of [Cu(MeCN)₄]PF₆ (0.02 Eq) and TBTA (Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, 0.02 Eq). The final product, named ZD2-Gd(HP-DO3A), was purified by RP-HPLC and lyophilized after overnight reaction. Relaxation times of aqueous solution of ZD2-Gd(HP-DO3A) with different concentration were measured at 1.5T using a Bruker minispec relaxometer at 37 °C. The T₁ and T₂ relaxivities were calculated from the slopes of the plots of 1/T₁ and 1/T₂ versus Gd concentrations. The structure of targeted MRI contrast agent, ZD2-Gd(HP-DO3A), is illustrated in Fig.1A. The contrast agent has relaxivities of r₁=5.29 mM^-1s^-1 and r₂=6.21 mM^-1s^-1 as calculated with the plot shown in Fig. 1B. MR imaging of PC3 tumor models indicated the preferential accumulation of ZD2-Gd(HP-DO3A) in tumor. As shown in Fig.1C and Fig.1D, ZD2-Gd(HP-DO3A) achieved a higher and sustained tumor accumulation compared with ProHance^® , resulting a 2-3 fold increase in CNR throughout 60 min after injection. We developed a targeted MRI contrast agent for molecular imaging of aggressive PCa. Our result showed an increased sensitivity for MRI detection of aggressive PCa using the EDB-FN targeted contrast agent, ZD2-Gd(HP-DO3A), compared with the clinical control agent ProHance^®. Further imaging studies are on-going to validate our imaging approach to differentiate PCa aggressiveness using ZD2-Gd(HP-DO3A). This contrast agent can potentially facilitate accurate risk stratification and clinical management of PCa.