Release Activated Iron Oxide Nanoparticles (REACTION) of Cellulose: A Magnetic Relaxation Switch for Environmentally sensitive MRI

INTRODUCTION: While magnetic relaxation switches (MRSws) have been developed using iron oxide nanocrystal cores that cluster together in the presence of an extracellular stimulus [Bogdanov, et al, Mol Imaging (1) 2002], the potential of using such systems to monitor intracellular events in vivo via MRI has not been investigated to date. Granot et al [Granot et al, ISMRM, 2009] first demonstrated an MRSw operating in cellulo and in vivo via MRI using a Feridex®/dextranase system. However, the dilute nature of Feridex labeling makes it a poor choice for any long-term tracking of intracellular processes via MRI. We hereby demonstrate the fabrication of magnetic cellulose particles that are relaxometrically sensitive to cellulase digestion. In the ‘off’ state, the particle remains intact. When switched ‘on’ through the cleavage action of cellulase, the iron oxide cores get dispersed, changing the relaxivity of the agent. MATERIALS AND METHODS: Fabrication of magnetic cellulose particles: 10-nm magnetite nanocrystals were synthesized via thermal decomposition of iron oleate. Magnetic cellulose particles were then fabricated using an oil-in-water single emulsion method previously described, using cellulose acetate as starting material [Nkansah et al, ISMRM, 2010]. Pure cellulose particles were regenerated via alkaline saponification. Iron content of particles was measured using ICP spectroscopy. Particle size and core distribution within particles were determined by SEM and TEM, respectively. Activity of cellulase on cellulose particles: Enzymatic activity of cellulase on cellulose particles was assessed by using a hexokinase-based system to measure the initial rate of glucose release from cellulose particles after a 2-hr treatment. In vitro study: For the in vitro study, magnetic cellulose particles (n=3) at a basis [Fe] of 2 mM were treated with 24 μm T.reesei/T.viride cellulase (pH 5.0, 0.05 M acetic acid) for 3 days at 37°C. Samples were then suspended in agarose gel phantoms at different iron concentrations. r2 and r2* relaxivity measurements were made at 4.7 T. Control samples were comprised of particles incubated only in acetate buffer or particles incubated with cellulase in DMEM cell growth medium (pH 7.4). In cellulo study: MCF-7 human cancer cells (n=3) were labeled for 3 days with DMEM containing magnetic cellulose particles (at basis [Fe] of 2 mM) and cellulase (24 μm) and used to make agarose gel phantoms. T2 and T2* relaxation times were then measured at 4.7 T. Control samples were cells incubated only with particles. RESULTS AND DISCUSSION: Cellulose particles were fabricated at a size of 414 ± 182 nm. Iron oxide nanocrystals were optimally loaded with a magnetite content of 70%. TEM showed good distribution of magnetite cores throughout the particles. Table 1 lists the r2 and r2* molar relaxivity of cellulosic nanoparticles, alongside those of inert MPIOs and dextran coated nanoparticles. Cellulose nanoparticles dissolved to produce glucose when treated with cellulase enzyme. The initial rate of glucose formation was found to obey classic Michaelis-Menten kinetics (Fig. 1). Magnetic cellulose particles subjected to a 3-day treatment with 1 mM cellulase at 37°C in 0.05 M acetic acid (pH 5) showed significant degeneration in morphology (Fig. 2a,b). Electron micrographs show what seem to be aggregates of amorphous cellulose fibers, left over from degradation. Furthermore, the reaction mix of the cellulase-treated group was much darker than control (Fig. 2c). Relaxometric measurements of the two reaction mixtures showed a clear difference in r2 relaxivity with the treatment group having 63% higher r2 than control with the disparity in r2* relaxivity not being as high, only 15% (Fig. 3). Because r2* values are so much higher than r2 for both treatment and control groups, we hypothesize that these particles exist in the static dephasing regime (where r2*>>r2) [Muller et al, MRM 22(2) 1991]. As such, a reduction in the effective size of magnetic cellulose particles in the static dephasing regime should trigger an increase in r2 with little effect on r2*. As cellulase acts on the particles, more iron cores get exposed to the surrounding medium, allowing for easier approach of water protons. A different situation was observed in cellulo, where cells labeled with both particles and enzyme exhibited lengthened T2 and T2* versus control cells labeled only with particles (Table 2). We interpret this to be due to recycling of the iron particles within the cells as they are released from the cellulose matrix, and hence, a loss in superparamagnetism. To prevent this, future modifications to these particles must include surface protection for the iron cores, such as gold coating. Nevertheless, these results are encouraging and demonstrate the potential of using environmentally sensitive agents to report on intracellular events using MRI. We believe, for example, that the differentiation of a transgenic neural progenitor cell line that expresses cellulase as a reporter gene when differentiating into a neuron could be visualized via MRI, if it is magnetically labeled using a cellulosic MRI agent like the one described here.