Vascular-targeted Magnetic Nanoparticles for Image-guided Cancer Therapy
Sudath Hapuarachchige1, Robert Ivkov2, and Dmitri Artemov1

1Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, United States, 2Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, United States


Bionized nanoferrites are magnetic nanoparticles, which can be used as contrast agents and therapeutic platforms for alternating magnetic field (AMF) induced hyperthermia. One important application is enhancing of vascular permeability in tumors for delivery of nanodrugs. We studied BNF nanoparticles specifically targeted to the tumor vasculature via VEGF receptors ligands. Targeted BNF particles were visualized by intravital multiphoton and MR imaging, and increased accumulation of targeted BNF was detected in breast cancer models.


Bionized nanoferrites (BNF) magnetic nanoparticles (MNP) possess unique capabilities as imaging agents for MRI and/or MPI as well as therapeutic agents for hyperthermia.1 However, at the current state of development, the effective delivery of BNF-MNP to target cancer cells still remains a key problem for successful imaging and therapeutic applications. While it is believed that the distribution and retention of BNF-MNPs in tumors can be enhanced by conjugating them with high affinity molecules, such as antibodies, antibody fragments, and peptides, practically no extravasation and tumor delivery of nanoparticles with diameters of about 100 nm beyond the perivascular space was detected in experimental models.2 In this study, we have demonstrated vascular targeting of single-chain VEGF (scVEGF) targeted BNF to the vasculature of triple-negative breast cancer models and suggesting that this approach could be used for temperature-based increase in vascular permeability for nano-delivery in cancer therapy (Fig 1).3


BNF-MNPs with aminated surface were labeled with rhodamine fluorophore (Rhod) and scVEGF by fast-reactive click chemistry and the excess amine groups in BNF surface were neutralized by PEG linkers (Fig 1) to prepare BNF-scVEGF-Rhod. Untargeted component, BNF-Rhod was prepared by labeling BNF with rhodamine and neutralizing excess amine groups by PEG linkers. Orthotopic MDA-MB-231/luc tumor mouse models were established by inoculating 2×106 cells dispersed in 50 μL of 50% Matrigel™/Hanks' balanced salt solution into the mammary fat pad of female athymic mice. The tumor uptake of targeted and untargeted MNPs was studied using BNF-scVEGF-Rhod and BNF-Rhod respectively. The intravital multiphoton fluorescence imaging was performed on custom-made mouse holder using Olympus FV1000MPE multiphoton laser-scanning microscope (Fig 2A). MRI studies were performed on a horizontal bore, preclinical 9.4T Bruker Biospec spectrometer using a home built single-turn solenoid coil. T2-weighted images of the tumors were acquired using the rapid acquisition with refocusing echoes (RARE) sequence before and after the intravenous administration of BNF-scVEGF at a dose of 50 μmol eq. Fe/kg, with a repetition time (TR) of 4 s and four effective echo times (TE; 7, 21, 35, and 49 ms) to locate the BNFs (isotropic field of view = 15 mm; matrix size of 128×128). In model hyperthermia experiments, we irradiated tumor and muscle capillaries using 800 nm laser beam of the microscope and observed the extravasation of fluorescent dextran-Rhod (70 kDa) and dextran-FITC (2 MDa) molecules (Fig 2B).


Increased extravasation of dextran-Rhod (70 KDa) and dextran-FITC (2 MDa) following local irradiation of the blood capillaries by NIR laser beam (800 nm) was detected in muscles (Fig 2B-i) and tumors (Fig 2B-ii), respectively. The estimated local temperature increase was approximately 10 0C (to 430C, 21 W/cm2). The target-specific BNF-scVEGF-Rhod shows high tumor uptake and accumulation in the tumor vasculature, visualized by dextran-FITC marker, compared to the untargeted BNF-Rhod (Fig 3). In MRI study, an increased accumulation of the components was detected for vascular-targeted BNF-scVEGF-Rhod compare to the untargeted BNF-Rhod (Fig 4).


The NIR irradiation of vasculature demonstrated the enhancement of extravasation of high molecular mass molecules into the tumor microenvironment presumably by thermal effect of endothelial cells.3 The study in tumor microenvironment was performed using an intravital multiphoton microscope. It is a powerful technique to study dynamic processes in living animals that enables one to observe cancer cells, tumor microenvironment, and microvascular architecture in vivo, in tumor models. A custom-made mouse holder that can fix orthotopic tumors for imaging without motional artifacts facilitated intravital imaging of cancer cells and vascular architecture. This model system suggests the future application of hyperthermia inducing MNPs for nano-delivery in tumors. Interestingly, muscle blood capillaries appeared significantly more resistant to the effects of NIR irradiation compared to the immature tumor vasculature. The targeted BNF-scVEGF-Rhod demonstrated increased the accumulation in tumors because of high expression levels of VEGF receptors in the neovascular endothelium. The effect was fast and observed within 1 min post-administration of targeted nanoparticles compared to the untargeted BNF-Rhod. The T2 imaging confirmed reliable MRI detection of the accumulation of targeted BNF-scVEGF-Rhod in tumor vasculature.


The vascular-targeted BNF nanoparticles have enhanced accumulation in the tumor vasculature and can be used for target-specific image-guided therapeutic applications.


We thank Mr. D. Jacob for his help with MP intravital microscopy; this study was supported by NIH R01CA154738.


(1) Dennis CL, Krycka KL, Borchers JA, et al. Internal magnetic structure of nanoparticles dominates time-dependent relaxation process in a magnetic field. Adv Funct Mater. 2015;25:4300-4311. (2) Kievit FM, Zhang M, Surface engineering of iron oxide nanoparticles for targeted cancer therapy. Acc Chem Res. 2011;44(10):853-862. (3) Bagley AF, Scherz-Shouval R, Galie PA, et al. Endothelial thermotolerance impairs nanoparticle transport in tumors. Cancer Res. 2015;75(16):3255-3267.


Fig 1. Schematics of the delivery of untargeted and targeted BNF-scVEGF-Rhod to VEGF-receptor overexpressing tumor vasculature and detailed structures of the targeted (BNF-scVEGF-Rhod) and untargeted (BNF-Rhod) nanaparticles.

Fig 2. (A) Custom-made mouse holder and intravital imaging setup. (B). Intravital MP images before (t=0) and 3 min (t=3 min) after laser irradiation of vascular capillaries. Extravasation of dextran-Rhod in muscle vasculature (i) and dextran-FITC in MDA-MB-231 tumor vasculature (ii).

Fig 3. Multiphoton microscopic images of BNF-scVEGF-Rhod and BNF-Rhod in the vasculature of MDA-MB-231 orthotopic models showing high accumulation of targeted BNF-scVEGF-Rhod compared to the untargeted BNF-Rhod.

Fig 4. MRI of systemically injected targeted BNF-scVEGF-Rhod (top panel) and untargeted BNF-Rhod (bottom panel) in orthotopic MDA-MB-231 tumor models showing high accumulation of targeted BNF-scVEGF-Rhod in tumor.

Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)