Nanomaterials provide large surface areas, relative to their volumes, on which to load functions. One challenge, however, has been to achieve precise control in loading multiple functionalities. Traditional bioconjugation techniques, which randomly target the surface functional groups of nanomaterials, have been found increasingly inadequate for such control--a drawback which may substantially slow down or prohibit the translational efforts. In the current study, we evaluated ferritin nanocages as candidate nanoplatforms for multifunctional loading. Ferritin nanocages can be either genetically or chemically modified to impart functionalities to their surfaces, and metal cations can be encapsulated in their interiors by association with metal binding sites. Moreover, different types of ferritin nanocages can be disassembled under acidic condition and reassembled at pH of 7.4, providing a facile way to achieve function hybridization. We were able to use combinations of these unique properties to produce a number of multifunctional ferritin nanostructures with precise control of their composition. We then studied these nanoparticles, both in vitro and in vivo, to evaluate their potential suitability as multimodality imaging probes. A good tumor targeting profile was observed, which was attributable to both the enhanced permeability and retention (EPR) effect and biovector mediated targeting. This, in combination with the generalizability of the function loading techniques, promises ferritin particles as a powerful nanoplatfom in the era of nanomedicine.Keywords ferritin nanocage; multimodality molecular imaging; positron emission tomography; near-infrared fluorescence imaging; integrin; RGD peptideThe idea of multimodality imaging has recently gained popularity 1, 2 . The rationale arises from the notion of improving the quality and accuracy of disease management by combining * To whom correspondence should be addressed, Shawn.Chen@nih.gov. ⊥ Both authors contributed equally to this work. Supporting InformationAdditional information regarding the expression and purification of Fn and R-Fn, preparation of C-Fn, synthesis of chimeric ferritin nanocages, cell binding assay, small animal PET and NIRF optical imaging studies, immunofluorescence staining, results of DLS analysis of R-Fn, and results of ex vivo imaging of excised tumors. This material is available free of charge via the Internet at http://pubs.acs.org. 6,7 . Such a transition to multimodal imaging poses a challenge to the design and synthesis of new generations of imaging probes. It requires that a targeting motif be integrated in a compact and controllable way with multiple imaging tags--something that is difficult to achieve with traditional biomaterials such as peptides and proteins. The emergence of nanotechnology, however, is expected to provide solutions to such challenges. With large ratios of surface-area-to-volume and multiple binding sites, nanomaterials can be loaded with a number of motifs [8][9][10] . However, the monotonicity of the...

The imaging of sentinel lymph nodes (SLNs), the first defense against primary tumor metastasis, has been considered as an important strategy for noninvasive tracking tumor metastasis in clinics. In this study, we report the development and application of mesoporous silica-based triple-modal nanoprobes that integrate multiple functional moieties to facilitate near-infrared optical, magnetic resonance (MR) and positron emission tomography (PET) imaging. After embedding near-infrared dye ZW800, the nanoprobe was labeled with T1 contrast agent Gd3+ and radionuclide 64Cu through chelating reactions. High stability and long intracellular retention time of the nanoprobes was confirmed by in vitro characterization, which facilitate long-term in vivo imaging. Longitudinal multimodal imaging was subsequently achieved to visualize tumor draining SLNs up to 3 weeks in a 4T1 tumor metastatic model. Obvious differences in uptake rate, amount of particles, and contrast between metastatic and contralateral sentinel lymph nodes were observed. These findings provide very helpful guidance for the design of robust multifunctional nanomaterials in SLNs’ mapping and tumor metastasis diagnosis.

An ongoing effort in the field of nanomedicine is to develop nanoplatforms with both imaging and therapeutic functions, the “nano-theranostics”. We have previously developed a human serum albumin (HSA) coated iron oxide nanoparticle (HINP) formula and used multiple imaging modalities to validate its tumor targeting attributes. In the current study, we sought to impart doxorubicin (Dox) onto the HINPs and to assess the potential of the conjugates as theranostic agents. In a typical preparation, we found that about 0.5 mg of Dox and 1 mg of iron oxide nanoparticles (IONPs, Fe content) could be loaded into 10 mg of HSA matrices. The resulting D-HINPs (Dox loaded HINPs) have a hydrodynamic size of 50 nm and are able to release Dox in a sustained fashion. More impressively, the HINPs can assist the translocation of Dox across cell membrane and even its accumulation in the nucleus. In vivo, D-HINPs retained a tumor targeting capability of HINPs, as manifested by both in vivo MRI and ex vivo immunostaining results. In a follow-up therapeutic study on a 4T1 murine breast cancer xenograft model, D-HINPs showed a striking tumor suppression effect that was comparable to Doxil and greatly outperformed free Dox. Such a strategy can be readily extended to load other types of small molecules, making HINP a promising theranostic nanoplatform.