R2 enhancement by formation of a tungsten-iron alloy crystal in the apoferritin cavity

V. Clavijo Jordan, and K. M. Bennett School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, United States Introduction: A fundamental challenge in molecular MRI is the sensitivity to delivered contrast agents (1). Thus, there is a critical need to increase the relaxivity of current contrast agents to detect sub-nanomolar concentrations in vivo. Natural nanoparticles are attractive agents due to its biodegradability and delivery properties (2). Ferritin, a 12nm iron carrier protein has been used as a natural contrast agent. However, the protein in its native form possesses a weakly magnetic crystal core that has a relaxivity of ~ 1-10mM-1s-1 Previous groups have developed various synthesis methods to increase its effectiveness and to load the protein core with highly magnetic iron oxide crystals (3, 4). In order to increase per-ion and per-particle relaxivity, one way of enhancing the magnetic properties for particles that are small enough to contain of a single magnetic domain, less than ~30nm, is to create an alloy of different magnetic metals (5). In this work we formed an alloy crystal in the interior of the apoferritin cavity in an effort to enhance R2 and increase the process yield. Although tungsten is diamagnetic, its inclusion in the crystal formed a formed a tungsten-iron alloy, with a per-particle relaxivity of 433,651mM-1s-1 and per-iron of 27,666mM-1s-1 and a percent yield increase of 200% compared to that of magnetoferritin. Methods: Particle Synthesis: A 2μM Apoferritin solution (Sigma Aldrich) was buffered in 0.05M MES at pH 8.5, 48mM FeCl2 (Sigma Aldrich) and 48mM sodium Tungstate Dihydrate (Sigma Aldrich) were de-aerated for 15 minutes with N2. The solution was kept at a temperature of 55 to 60°C. We added 125μl of FeCl2 to the apoferritin solution every 10 minutes for a total of 20 additions, after the 10th addition 125μl of Sodium tungstate was added every 5 minutes after every FeCl2 addition. Samples were dialyzed against 0.15M NaCl, and filtered using a magnetic column (Miltenyi Biotec), and eluted into 0.15 NaCl buffer. As a protein control, 2μM bovine serum albumin (Thermo Scientific) was used instead of apoferritin. Total protein concentration was obtained with a Bradford assay, and inductively coupled plasma – optical emission spectroscopy (ICP-OES) was used to measure metal concentrations. Relaxometry: The particle relaxivity was measured using a 1.5T Bruker Minispec relaxometer. Bruker’s curve-fitting tool was used to find the corresponding T2 values (Inter-pulse τ = 10ms, 200 points) and T1 values (pulse separations ranging from 5 to 20000ms, 4 scans, 10 points) of samples suspended in a 1% agarose gel. Electron Microscopy: Particle samples were adsorbed on Cu-C grids and transmission electron microscopy (TEM) images were obtained using a Philips CM12 electron microscope. High Resolution Electron Microscopy (HREM) images were obtained using a Philips CM200-FEG TEM/STEM. Electron Spin Resonance: EPR was performed with a X-band spectrometer (Bruker ESP300E) with 5mW power, 25G modulation and at a temperature of 5K under liquid helium. Results and Conclusions: Loading the apoferritin core with an alloy of tungsten and iron resulted in an increased per-iron and per-particle relaxivity (R2) of 27,666mM-1s-1 and 433,651mM-1s-1 respectively, (see Figure 1). This synthesis procedure along with the addition of a diamagnetic metal increased the nanoparticle yield after filtration by 200% when compared to magnetoferritin. Also, ICP-OES indicated that ~724 Fe ions and 7,454 tungsten ions are present within the protein. TEM showed the formation of electron dense metallic cores of mixed composition with diameters ranging from 5-7.5nm which are larger than native ferritin and magnetoferritin (Figure 2). HREM also showed that the crystal structures in the core are formed in a multi-twinned fashion each direction with lattice spacing of 2.5Å corresponding to magnetite (Figure 3). Electron spin resonance showed that the newly synthesized W-Fe alloy nanoparticles had less Fe(III) in its cores compared to magnetoferritin. The presence of Fe(III) in the cores was confirmed by the typical iron peak at g=4.3. By contrast, FeCl2 (a Fe(II) state) did not show paramagnetic signal in the spectrum (Figure 3). We conclude that the magnetic properties (R2) of magnetoferritin and the % yield can be strongly enhanced by the addition of a diamagnetic metal into the synthesis to form an alloy crystal in the apoferritin cavity.