15 16 17 18 19 20 21 22 23 24 25 ABSTRACT26 Nanoparticles often only exploit the upregulation of a receptor on cancer cells to enhance 27 intratumoral deposition of therapeutic and imaging agents. However, a single targeting moiety 28 assumes that a tumor is homogenous and static. Tumoral microenvironments are both 29 heterogenous and dynamic, often displaying variable spatial and temporal expression of 30 targetable receptors throughout disease progression. Here, we evaluated the in vivo performance 31 of an iron oxide nanoparticle in terms of targeting and imaging of orthotropic mouse models of 32 aggressive breast tumors. The nanoparticle, a multi-component nanochain, was comprised of 3-33 5 iron oxide nanoparticles chemically linked in a linear chain. The nanoparticle's surface was 34 decorated with two types of ligands each targeting two different upregulated biomarkers on the 35 tumor endothelium, P-selectin and fibronectin. The nanochain exhibited improved tumor 36 deposition not only through vascular targeting but also through its elongated structure. A single-37 ligand nanochain exhibited a ~2.5-fold higher intratumoral deposition than a spherical 38 nanoparticle variant. Furthermore, the dual-ligand nanochain exhibited higher consistency in 39 generating detectable MR signals compared to a single-ligand nanochain. Using a 7T MRI, the 40 dual-ligand nanochains exhibited highly detectable MR signal within 3h after injection in two 41 different animal models of breast cancer. 42 43 44 45 KEYWORDS 46 Iron oxide nanochains; dual-ligand nanoparticle; breast cancer, MRI 47 48 49 50 51 52 53 54 INTRODUCTION
55Imaging is critical for management of patients with breast cancer including diagnosis, 56 treatment planning and response assessments. To improve cancer imaging, various targeting 57 schemes have been employed to direct nanoparticle imaging agents to cancers [1, 2]. Traditional 58 targeting strategies decorate the surface of nanoparticles with a ligand directing them to 59 upregulated receptors on breast cancer cells within the tumor interstitium. Rather than targeting 60 the tumor interstitium, an alternative strategy is to use vascular targeting and direct the 61 nanoparticles to the altered endothelium associated with breast cancer. The endothelium of 62 tumors, including those of the breast, displays a wide variety of targetable biomarkers that are not 63 readily found on healthy endothelium. For circulating nanoparticles, the endothelium is the closest 64 point-of-contact, which facilitates direct access to the targetable vascular biomarkers of the 65 disease [3-9]. Considering their size and multivalent avidity, nanoparticles are ideal for vascular 66 targeting.
67Further, the shape of the nanoparticle can dictate its targeting avidity [7,[10][11][12]. Recently, 68 we reported a new one-pot synthetic concept for making multicomponent chain-like nanoparticles 69 (termed nanochains), which are comprised of about three iron oxide nanospheres chemically 70 linked into a linear, chain-like assembly [13]. ...