Design, synthesis and biological evaluation of ultrasmall iron oxide nanoparticles for cancer imaging

There is currently a great interest in the application of nanoparticles for molecular imaging [1]. This results in the development of both, organic and inorganic nanoparticles functionalized in a way that radionuclides, targeting ligands, and different biopolymers can be attached in order to provide an imaging signal and alter the pharmacokinetic properties [2]. Ultrasmall supraparamagnetic iron oxide (Fe3O4) nanoparticles (USPIOs) are one of the most widely studied nanomaterials. USPIOs possess unique magnetic properties that make them attractive candidates as advanced biomedical materials [3]. Furthermore, the biocompatibility and favourable pharmacokinetic profile of USPIOs makes them suitable to be used as multifunctional agents. They can serve as contrast agents for clinical use in magnetic resonance imaging (MRI), positron emission tomography (PET) and optical imaging (OI) [4]. One strategy to improve retention of nanoparticles in tumours and accumulation in cancer cells is active targeting to specific cell membrane receptors. The epidermal growth factor receptor (EGFR) is overexpressed in more than 30% of all epithelial cancers [5]. With a wide clinical application of EGFR-targeted therapy, our effort is focused on obtaining magnetic nanoparticles to target EGFR for early diagnosis. This work aims at the development of new nanotracers based on USPIOs coated with a biodegradable polymer that are also decorated with agents for radiochemical and/or fluorescence imaging and EGFR-specific ligands as targeting units.
Fig 1: (A) Schematic representation of the crystal structure magnetite and (B) TEM image of iron oxide nanoparticles. The production of hydrophobic USPIOs has been achieved using thermal decomposition of metal-oleate precursors. These reaction of metal chlorides and sodium oleate. The subsequent precipitation procedure gives monodisperse nanocrystals within a size of 4-5 nm as it shown figure 1B [8]. The main limitation of using USPIOs for in vivo applications is their instability under physiological conditions. Different biocompatible polymers such as polyvinylalcohol, polyacrylic acid have been tested for the hydrophilic coating of the iron oxide core to make them stable in aqueous solution [9]. After an intensive physicochemical characterization including studies of their stability in water and several buffers, the carboxy methyl polyvinyl alcohol (CMPVA) was chosen as the most suitable coating agent for the surface modification of USPIOs [10]. The stability of the USPIOs coated with CMPVA was studied in water and different buffer solutions, where it was found that they were stable up to two months. Figure 2 shows the size distribution using DLS (dynamic light scattering, Zetasizer Malvern) of a representative sample and its zeta potential in water.
Fig 2: (A) DLS size by number (8±2nm) and (B) Zeta potential (-35mV) of coated CMPVA-USPIOs. Our first goal was to engeneer and functionalize the surface of the hydrophilic USPIOs. This included the attachment of the fluorescent label BODIPY to the CMPVA shell. This enabled the USPIOs to be suitable for in vitro binding and uptake studies in cancer cells by fluorescence imaging. Furthermore, preliminary radiolabeling studies (64Cu) have been undertaken using USPIOs decorated with functional copper chelating agents, such as NOTA. Radio-HPLC and Radio-TLC have been applied to get information about the labelling efficiency and stability of the 64Cu-labeled USPIOs. From our point of view, the generated nanoparticles possess promising features as novel imaging agents for in vivo cancer diagnostics. Experiments concerning the bioconjugation of EGFR-specific peptides on the surface of USPIOs are currently underway.

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