Coating optimization of superparamagnetic iron oxide nanoparticles for high T2 relaxivity

Abstract

We describe a new method for coating superparamagnetic iron oxide nanoparticles (SPIOs) and demonstrate that, by fine-tuning the core size and PEG coating of SPIOs, the T2 relaxivity per particle can be increased by >200-fold. With 14 nm core and PEG1000 coating, SPIOs can have T2 relaxivity of 385 s-1 mM-1, which is among the highest per-Fe atom relaxivities. In vivo tumor imaging results demonstrated the potential of the SPIOs for clinical applications.

Figure 1. A schematic diagram of SPIO synthesis and coating schemes

a. Both iron oxide cores and DSPE-PEG are dissolved in chloroform. b. Addition of DMSO induces assembly between iron oxide cores and DSPE-PEG molecules. c. Transition into water further strengthens the hydrophobic interaction between DSPE-PEG and oleic acid/oleylamine on iron oxide cores. Due to its extremely low CMC (~5µM in water), unoccupied DSPE-PEG exists mainly in the form of empty micelles. d. A schematic diagram of a SPIO with 4.8nm iron oxide core and DSPE-mPEG1000 coating. 1 through 5 represent PEG, phosphate, DSPE, oleic acid / oleylamine and the iron oxide core, respectively. The dimensions are based on TEM measurement and numerical analysis (supplementary information, SII & SIII). e. Dynamic light scattering of the SPIOs coated with DSPE-mPEG1000. Blue: Number-weighted size distribution of 5nm iron oxide core coated with DSPE-mPEG1000, average size = 14.8±1.2 nm. Purple: 14 nm iron oxide core coated with DSPE-mPEG1000, average size = 28.6±0.4 nm. Shown in the inset are TEM images of iron oxide cores and coated SPIOs negatively stained with phosphotungsic acid to give a white layer surrounding the iron oxide cores indicating the DSPE-mPEG1000 coating layer.

Figure 2. Dependency of T₂ relaxivity on the core size and the PEG chain length of the SPIOs

a. T₂ relaxivity of the SPIOs with constant iron concentration. b. T₂ relaxivity of the SPIOs on a per particle basis. SPIOs with two core sizes, 5nm and 14nm, and five PEG sizes, molecular weight of 550, 750, 1000, 2000 and 5000 Da, were evaluated. c. Normalized magnetic field of the two iron oxide cores (Left: 5nm & Right 14nm). The color bar represents the magnitude of the magnetic field strength (z-component) of the iron oxide cores. The magnetic field strength is normalized by its value at the equator line on the core surface. Solid line and dashed lines represent the boundaries of lipid bilayer and PEG coating layers, respectively. Starting from the centre, the dash lines indicate PEG550, PEG750, PEG1000, PEG2000 and PEG5000.

Figure 3. In vitro targeting

A 96-well ELISA plates were coated with mouse IgG at designated protein loading concentration. The wells coated with mouse IgG was incubated with either antibody conjugated horseradish peroxidase or the SPIOs at 37°C for 1 hour. a. Wells were incubated with goat anti-mouse IgG conjugated with horseradish peroxidase. Horseradish peroxidase activity was detected by ABTS. b. Wells was incubated with SPIOs conjugated with goat anti mouse IgG. Iron content of bound SPIOs was measured using Ferrozine method. c and d. The plate was loaded with designated amount of 5nm or 14nm SPIOs suspended in 50µl of water and imaged with a 7T MRI instrument using spin-echo sequence with echo time equal to 12ms and 60ms, respectively. e. T₂ effect calculated based on MRI images.

Figure 4. In vivo tumor imaging

MRI experiments were performed using spin-echo sequence. a. Arrow shows the location of the subcutaneous tumor. b and c. MR images of tumor before probe injection. d and e. MR images collected after 1 hour following the injection of 14nm SPIOs conjugated with antibodies against mouse VEGFR-1. Red dotted lines in b and d outline the tumor. Scale bar represents 5 mm.