A molecular receptor targeted, hydroxyapatite nanocrystal based multi-modal contrast agent

Abstract

Multi-modal molecular imaging can significantly improve the potential of non-invasive medical diagnosis by combining basic anatomical descriptions with in-depth phenotypic characteristics of disease. Contrast agents with multifunctional properties that can sense and enhance the signature of specific molecular markers, together with high biocompatibility are essential for combinatorial molecular imaging approaches. Here, we report a multi-modal contrast agent based on hydroxyapatite nanocrystals (nHAp), which is engineered to show simultaneous contrast enhancement for three major molecular imaging techniques such as magnetic resonance imaging (MRI), X-ray imaging and near-infrared (NIR) fluorescence imaging. Monodispersed nHAp crystals of average size ∼30 nm and hexagonal crystal structure were in situ doped with multiple rare-earth impurities by a surfactant-free, aqueous wet-chemical method at 100 °C. Doping of nHAp with Eu 3+ (3 at%) resulted bright near-infrared fluorescence (700 nm) due to efficient 5 D 0– 7 F 4 electronic transition and co-doping with Gd 3+ resulted enhanced paramagnetic longitudinal relaxivity ( r 1 ∼12 mM −1 s −1) suitable for T 1 weighted MR imaging together with ∼80% X-ray attenuation suitable for X-ray contrast imaging. Capability of MF-nHAp to specifically target and enhance the signature of molecular receptors (folate) in cancer cells was realized by carbodiimide grafting of cell-membrane receptor ligand folic acid (FA) on MF-nHAp surface aminized with dendrigraft polymer, polyethyleneimine (PEI). The FA-PEI-MF-nHAp conjugates showed specific aggregation on FR +ve cells while leaving the negative control cells untouched. Nanotoxicity evaluation of this multifunctional nHAp carried out on primary human endothelial cells (HUVEC), normal mouse lung fibroblast cell line (L929), human nasopharyngeal carcinoma (KB) and human lung cancer cell line (A549) revealed no apparent toxicity even upto relatively higher doses of 500 μg/mL and 48 h of incubation. Flow-cytometry based reactive oxygen species (ROS) analysis also showed no significant levels of ROS generation in the nHAp treated cells. The tri-modal contrast imaging functionality together with molecular receptor targeting capability and biocompatibility makes MF-nHAp a promising biomineral contrast agent for combinatorial molecular imaging.