Recently, we have shown that small molecular targeted MRI contrast agents specific to biomarkers abundant in tumor extracellular matrix is effective to produce strong signal enhancement for detection of primary tumors and micrometastases in animal models.^{1, 2} Small molecular MRI contrast agents are not effective to detect the biomarkers expressed on cancer cells because of the low concentration of the biomarkers and low sensitivity of MRI.³ To produce MRI detectable signal enhancement for the biomarker on cancer cell surface, we developed a targeted host-guest nanosized contrast agent cRGD-POSS-βCD-(DOTA-Gd)-Cy5. cRGD (cyclic Arg-Gly-Asp-(D)Phe-Lys) is a tumor homing cyclic peptide that specifically bind to α_vβ₃ integrin on tumor vascular endothelial cells and as well as various tumor cells, such as mammary carcinoma cells.⁴ The tumor homing peptide cRGD, macrocyclic Gd(III) chelates (DOTA-Gd) and Cy5 fluorescent probes were loaded on the nanosized carrier by host-guest interaction to develop a facile and customizable nanosized contrast agent by host-guest interaction for targeted molecular imaging of breast cancer.The targeted host-guest contrast agent cRGD-POSS-βCD-(DOTA-Gd)-Cy5 was synthesized by self-assembly of β-cyclodextrin (βCD) conjugated polyhedral silsesquioxane (POSS-βCD) and adamantane(Ad)-functionalized imaging agents and a targeting agent via the host-guest interaction. The effectiveness of the obtained contrast agent RGD-POSS-βCD-(DOTA-Gd)-Cy5 was demonstrated in a mouse 4T1 breast cancer model. Mice bearing 4T1-GFP-Luc2 breast tumors on flank were studied at 2-3 weeks post tumor implant. MRI study was performed using a Bruker Biospec 7 T MRI scanner (Bruker Corp., Billerica, MA, USA) with a volume RF coil. Mice were injected with cRGD-POSS-βCD-(DOTA-Gd)-Cy5 or the non-targeted contrast agent cRAD-POSS-βCD-(DOTA-Gd)-Cy5 at a dose of 0.1 mmol-Gd³⁺/kg (0.88 mmol Cy5/kg), respectively. Mice were anesthetized with a 2% isoflurane-oxygen mixture in an isoflurane induction chamber. Fat suppression 3D FLASH and T1-weighted 2D axial images were then acquired at different time points after the injection for up to 30 min. After MRI and 4 h post-injection of the agents, mice were sacrificed and tumors and major organs were collected and imaged by Maestro FLEX In Vivo Imaging System. Tumor slices were stained with a rabbit monoclonal anti-integrin α_v antibody antibody (ab179475, Abcam) or anti-integrin β₃ antibody (ab75872, Abcam), followed by Rhodamine-Red-X conjugated goat anti-rabbit IgG (H + L) (Jackson Immuno Research Lab, West Grove, PA). The slides were imaged by Olympus FV1000 confocal laser scanning microscope.Here, we used a nanosized carrier loading with multiple imaging agents thus to produce detectable signal enhancement for the α_vβ₃ integrin on cancer cell surface (Fig.1). The r₁ relaxivity of Ad-(DOTA-Gd) was 3.17 mM^-1s^-1 per Gd, measured at 1.5 T and 37 ^oC, comparable with reported values for clinical used small molecular contrast agents. The r₁ relaxivity increased to 6.36 mM^-1s^-1 per Gd after its complexation with βCD to form βCD-(DOTA-Gd). The r₁ value further increased to 9.50 mM^-1s^-1 per Gd when Ad-(DOTA-Gd) was complexed with POSS-βCD to form POSS-βCD-(DOTA-Gd). The target contrast agent cRGD-POSS-βCD-(DOTA-Gd)-Cy5 showed much strong tumor enhancement than ProhanceÒ and the nontargeted agent (Fig.2). The nanosized contrast agent was able to deliver a sufficient amount of Gd-DOTA chelates to its molecular target for effective tumor molecular imaging with MRI. cRGD-POSS-βCD-(DOTA-Gd)-Cy5 also provided strong fluorescence enhancement in tumor tissue than that of the non-targeted agent (Fig.3). cRGD-POSS-βCD-(DOTA-Gd)-Cy5 can provide strong contrast enhancement to delineate malignant tumors during molecular MR and fluorescent imaging. The α_v or β₃ immunostaining of tumor slice indicates specific binding of cRGD-POSS-βCD-(DOTA-Gd)-Cy5 to α_vβ₃ in tumor tissue (Fig.4). The method to prepare host-guest contrast agent, cRGD-POSS-βCD-(DOTA-Gd)-Cy5, may be an appropriate method to developing effective multi-modal imaging agent promising for clinical use.Molecular MRI and fluorescent imaging with cRGD-POSS-βCD-(DOTA-Gd)-Cy5 is promising for effective detection of breast cancer by targeting to the overexpressed α_vβ₃ on cell surface.