Ferrite-based magnetic nanoparticles (MNPs) have attracted great attention in different biomedical applications, mainly due to their attractive biological and magnetic properties [1], [2]. The intrinsic magnetic properties of NPs such as single-domain superparamagnetic behavior, high saturation magnetization values, and negligible coercivity and remnant magnetization have a prominent effect on the success of their biological applications [3]. These properties make MNPs ideal candidates for targeted drug delivery, magnetic resonance imaging (MRI), and magnetic hyperthermia [4], [5], [6], [7]. Among various MNPs, cobalt ferrite nanoparticles (CoFe NPs) have beenextensively studied due to their superior magnetic and enzymatic properties [8], [9], [10]. However, development of simple and efficient methods for synthesis and surface functionalization of CoFe NPs with improved physical and biological properties, suitable surface coating, and different functional groups will increase their applications [11].

A wide variety of organic and inorganic compounds have been used for surface coating of MNPs to improve their biocompatibility and colloidal stability, as well as to decrease their potential agglomeration and oxidation [12]. Recently, several reports have been published on the surface coating of MNPs with casein, as a biocompatible and biodegradable protein. The iron oxide cores with a casein shell has been reported to represent appropriate surface functionality with minimal adverse effects on their magnetic activity [13]. The casein coating can stabilize MNPs and improve their dispersity, biodistribution and bioactivity. Therefore, casein was used as the stabilizing agent in the synthesis of CoFe NPs.

The targeted nanocarriers capable of controlled drug delivery to specific organ or tissue have recently gained significant attention for achieving maximal therapeutic efficiency with minimal side effects [14], [15], [16], [17]. Different ligand molecules, such as antibodies, peptides, carbohydrates, and aptamers, are commonly used to target nanocarriers [18], [19]. Among these, antibodies and antibody fragments are the most commonly utilized ligands in personalized medicine for the precise diagnosis and effective therapy of disease [20]. Bevacizumab (Avastin®) is a monoclonal antibody used therapeutically for different cancers including colon, lung, ovarian, glioblastoma, hepatocellular carcinoma, and renal-cell carcinoma [21].

The suppression of angiogenesis, a key hallmark of cancer, is an important and efficient therapeutic strategy for cancer treatment [22]. Among different angiogenesis inhibitors, tyrosine kinase inhibitors (TKIs) are well-known and widely used in cancer therapy [23]. Sunitinib, a small molecule TKI, specifically blocks multiple tyrosine kinas receptors, resulting in anti-tumor and anti-angiogenic effects [24]. However, due to its different side effects, such as skin reactions, fatigue, diarrhea, and hypertension, controlled delivery via targeted nanocarriers is often recommended [25].

In the present study, CoFe NPs were hydrothermally synthesized in the presence of casein (CA), and the resulting casein-coated CoFe NPs (CACoFe NPs) were fully characterized. Subsequently, the CACoFe NPs were functionalized with bevacizumab antibody and loaded with sunitinib. These modified NPs were evaluated as a novel theranostic agent for cancer therapy and imaging (Scheme 1). The anticancer activity of the NPs was assessed by MTT assay, and their mechanism of action was further elucidated by the real-time polymerase chain reaction (PCR), flow-cytometry and Enzyme-linked Immunosorbent Assay (ELISA). Additionally, the potential of the NPs to trap vascular endothelial growth factor (VEGF) and their anti-angiogenic effect were evaluated in situ, in vitro, and in vivo. The ability of CACoFe NPs as a MRI contrast agent was also investigated both in vitro and in vivo.