Imaging breast cancer using a dual-ligand nanochain particle

Abstract

Nanoparticles often only exploit the upregulation of a receptor on cancer cells to enhance intratumoral deposition of therapeutic and imaging agents. However, a single targeting moiety assumes that a tumor is homogenous and static. Tumoral microenvironments are both heterogenous and dynamic, often displaying variable spatial and temporal expression of targetable receptors throughout disease progression. Here, we evaluated the in vivo performance of an iron oxide nanoparticle in terms of targeting and imaging of orthotropic mouse models of aggressive breast tumors. The nanoparticle, a multi-component nanochain, was comprised of 3-5 iron oxide nanoparticles chemically linked in a linear chain. The nanoparticle's surface was decorated with two types of ligands each targeting two different upregulated biomarkers on the tumor endothelium, P-selectin and fibronectin. The nanochain exhibited improved tumor deposition not only through vascular targeting but also through its elongated structure. A single-ligand nanochain exhibited a ~2.5-fold higher intratumoral deposition than a spherical nanoparticle variant. Furthermore, the dual-ligand nanochain exhibited higher consistency in generating detectable MR signals compared to a single-ligand nanochain. Using a 7T MRI, the dual-ligand nanochains exhibited highly detectable MR signal within 3h after injection in two different animal models of breast cancer.

Fig 1. Synthesis and characterization of the nanochain particles.

(a) Illustration shows the dual-ligand nanochain particle. (b) Reaction scheme shows the synthesis of nanochains using parent iron oxide nanoparticles with different surface functionality. (c) The size of the parent nanoparticles and nanochains was measured using dynamic light scattering (DLS). (d) TEM image of nanochain particles is shown. (e) The transverse (R2) relaxivity of the nanochains was measured at 1.5 Tesla using a relaxometer.

Fig 2. Organ distribution of targeting variants of spherical or chain-like iron oxide nanoparticles.

Mice were systemically injected with nanoparticles at a dose of 10 mg/kg Fe. The dual-ligand nanochain was compared to non-targeted nanochain, single-ligand nanochain targeting P-selectin and single-ligand spherical nanoparticles targeting P-selectin (n = 5 mice per formulation). Animals were euthanized 3 h after injection and organs were extracted and homogenized. The relaxation of the homogenate of different organs was measured using a 1.5 T Bruker Minispec and was recorded as tissue relaxivity per gram of tissue.

Fig 3. Comparison of tumor targeting accuracy of different targeted nanoparticles.

(a) Single-ligand nanochains targeting P-selectin and their spherical counterparts were IV injected in mice bearing mammary D2.A1 tumors. All formulations were administered at the same dose (10 mg/kg Fe). The animals were perfused 3 h after injection and the tumors were excised, homogenized and measured using a 1.5 T Bruker Minispec (data are represented as mean ± s.d.; n = 5 mice in each group; unpaired t-test, P values: ****<0.0001). (b) The endogenous relaxation rate (R2) of healthy mammary tissue were compared to those of tumor bearing mice after systemic injection of single-ligand or dual-ligand nanochains (data are represented as mean ± s.d.; n = 5 mice in each group; unpaired t-test, P values: **<0.005). (c) Quantification of the bioluminescent signal indicated similar tumor burden of the different groups of animals. (d) Z-score analysis was performed to identify the belongingness of each tumor bearing mouse injected with a formulation to a healthy mammary population. If the z-score probability value was larger than an alpha of 0.05, it signified that it was likely that the R2 value belonged to that of a healthy mammary tissue as opposed to a diseased mammary.

Fig 4. MR imaging of mice bearing mammary breast tumors using dual-ligand nanochains and a 7T MRI.

(a) Animals with 4T1 or D2.A1 mammary tumors were imaged before and 3 h after tail vein injection of dual-ligand nanochains at a dose of 10 mg/kg Fe. The signal intensity was measured in the tumor and healthy mammary fat pad. The signal intensity normalized to the signal of the healthy mammary tissue (scale: 0–1). The normalized values of 0 and 1 correspond to maximum and minimum contrast, respectively, compared to the pre-injection values of healthy mammary tissue (data are represented as mean ± s.d.; n = 3 mice for each animal model; unpaired t-test, P values: *<0.05, **<0.005). (b) Representative sagittal T2-weighted images of a mouse bearing a D2.A1 mammary tumor before and 3 h after injection of dual-ligand nanochains.

Fig 5. Histological evaluation of the expression of vascular biomarkers and intratumoral deposition of targeted nanoparticles in the orthotopic D2.A1 mouse model.

(a) Mice bearing mammary D2.A1 tumors were euthanized 3 h after tail vein injection of dual-ligand nanochains at a dose of 10 mg/kg iron. After perfusion, tumors were excised and processed for histology. Images from serial tissue sections show that topology of fibronectin and P-selectin with respect to the endothelium (10x magnification; nuclear stain: blue; cancer cells: green; CD31 endothelial marker or fibronectin or P-selectin: red; overlay: yellow). (b) The near-perivascular deposition of the dual-ligand nanochain was identified through an iron stain (left panel: 10x magnification; nuclear stain: blue and CD31 endothelial marker: red; middle and right panels: 20x magnification; iron stain: Prussian blue).