Introduction

Lanthanide metals except gadolinium (Gd) have highly anisotropic electron spin structure, their electron relaxation rates are on the order of ~10-13 s^-1. This anisotropic electron structure results in the short electron relaxation time (T_1e), molecular tumbling correlation time (τ_R) is always larger than T_1e. The curie relaxation mechanism becomes important on this account, it cause to increase transverse relaxation shortening effect especially with a high magnetic moment (μ_eff) and external field. Dysprosium (Dy) has the largest μ_eff (10.6 μB) among lanthanides, therefore, it can be used a useful low molecular T₂ MRI contrast agent (CA) in ultra-high field. We synthesized Dy complex (Dy(DO3A-ph-NH₂)) encapsulated apoferritin for use as a T₂ MRI CA for ultra-high field MRI.

Materials and Methods

All reagents were purchased from commercial sources and used as received. Characterization of the synthesized compounds have been determined by analytical spectroscopic methods.^{1, 2} The final paramagnetic composition concentration of Apo[Dy(DO3A-ph-NH₂)] was measured by ICP mass. T₁ measurements were carried out using an RARE-inversion recovery method with variable inversion times (TI) in various external fields (3 - 15.2 T). For T₂, the multi-slice multi-echo (MSME) sequence was adapted in same the external fields. R₁ and R₂ relaxation times were obtained from the nonlinear least-squares fit of the mean pixel values at variable TI and TE, respectively. The relaxivities (r₁ and r₂) were then calculated as a slope of linear fit of relaxation rate along with concentrations. The in vivo T₂ weighted MR image was obtained at 9.4 T animal MRI equipment (Agilent, U.S.). In these studies, the mice (25 ~ 27 g) were anesthetized by 1.5% isoflurane in oxygen. MR images were acquired before and after intravascular injection of Apo[Dy(DO3A-ph-NH₂)] (dose: 0.3 mmol Dy/kg). Also image parameters of axial images for fast spin-echo are as follows: Repetition time (TR) = 4000 msec; echo time (TE) = 12 msec; 30 30 mm field of view (FOV); 128×128 matrix size; 1.0 mm slice thickness; echo train length = 4; scan time of each image = 8 min 40 sec.

Results and Discussion

Low molecular Dy complex was synthesized according to precede literature1, the purity was characterized with FAB mass spectroscopy and HPLC analysis. Encapsulation of apoferritin was proceeded carefully by adjusting pH (pH 2.0-7.4)². After encapsulation, morphology of protein was well maintained in TEM images (figure 1). The absorbance change of Apo[Dy(DO3A-ph-NH₂)] was observed at 300-400 nm because of encapsulated Dy(DO3A-ph-NH₂) (figure 1). The r₂ relaxivities were considerably increased up to 69.63 mM^-1s^-1 at 15.2 T. And the r₂/r₁ ratio per Dy increased from 3.82 at 3 T to 55.26 at 15.2 T. (figure 2). The curie relaxation is affected by molecular tumbling correlation time (τ_R) and water residence time (τ_M). The preceding results from large molecular weight (~480 kDa) and restricted water access by protein out-sphere that respectively increase τ_R and τ_M. The concentration of Apo[Dy(DO3A-ph-NH₂)] for in vivo test were prepared to 44 mM per Dy from ICP-spectrometer measurement. The MR images of Apo[Dy(DO3A-ph-NH₂)] were acquired at 9.4T with liver tumor mice (figure 3). This senseful negative enhancement in liver shows the effect of T₂ MRI contrast agent at ultra-high field.

Conclusion

In conclusion, we have successfully synthesized Apo[Dy(DO3A-ph-NH₂)] as a T₂ MRI CA for ultra-high field MRI. Due to the high increment of r₂/r₁ ratio of Apo[Dy(DO3A-ph-NH₂)] above 9.4 T, we expected that it can be used as biocompatible negative-enhancing CA replacing metal-oxide based T₂ MRI CAs.

Acknowledgements

We gratefully acknowledge financial support from the Basic Science Research Program (NRF-2017R1D1A1B03031640) and BK21 Plus KNU Biomedical Convergence Program.