Kinetically inert manganese (II)-based hybrid micellar complexes for magnetic resonance imaging of lymph node metastasis

Abstract

The localization and differential diagnosis of the sentinel lymph nodes (SLNs) are particularly important for tumor staging, surgical planning and prognosis. In this work, kinetically inert manganese (II)-based hybrid micellar complexes (MnCs) for magnetic resonance imaging (MRI) were developed using an amphiphilic manganese-based chelate (C18-PhDTA-Mn) with reliable kinetic stability and self-assembled with a series of amphiphilic PEG-C18 polymers of different molecular weights (C18En, n = 10, 20, 50). Among them, the probes composed by 1:10 mass ratio of manganese chelate/C18En had slightly different hydrodynamic particle sizes with similar surface charges as well as considerable relaxivities (∼13 mM^-1 s^-1 at 1.5 T). In vivo lymph node imaging in mice revealed that the MnC MnC-20 formed by C18E20 with C18-PhDTA-Mn at a hydrodynamic particle size of 5.5 nm had significant signal intensity brightening effect and shortened T₁ relaxation time. At an imaging probe dosage of 125 μg Mn/kg, lymph nodes still had significant signal enhancement in 2 h, while there is no obvious signal intensity alteration in non-lymphoid regions. In 4T1 tumor metastatic mice model, SLNs showed less signal enhancement and smaller T₁ relaxation time variation at 30 min post-injection, when compared with normal lymph nodes. This was favorable to differentiate normal lymph nodes from SLN under a 3.0-T clinical MRI scanner. In conclusion, the strategy of developing manganese-based MR nanoprobes was useful in lymph node imaging.

Keywords: T1 contrast agent; amphiphilic manganese chelate; kinetically inert; magnetic resonance imaging; sentinel lymph node.

Graphical abstract

Figure 1.

Characterization of manganese-based hybrid micellar complexes (MnCs). (A) Hydrodynamic particle diameters of MnC-10, MnC-20, MnC-50, MnC-127 and micelles formed by single type of PEG-based polymers (C18E10, C18E20, C18E50 and F127). (B) Size variation of the four MnCs in seven days. (C) Zeta potential of the four MnCs before and after chelation with manganese. (D) TEM images and particle size distribution calculated by Image J. (E) EDS images of the four MnC formulations.

Figure 2.

Evolution of longitudinal relaxation rates R₁^p (t)/R₁^p (0) as a function of time for the four MnCs in the presence of Zn²⁺ or EDTA which is 1.5, 3 or 6 times than manganese ion at pH = 7.4, or in the environment of excessive hydrogen protons at pH = 5.0.

Figure 3.

Grayscale T₁-weighted and T₂-weighted MR images at 1.5 T of MnC-10, MnC-20, MnC-50, MnC-127 and the commercial gadolinium-based contrast agent, Gd-DOTA. (TE = 13.9 ms, TR = 90 ms, for T₁-weighted SE sequences and TE = 75 ms, TR = 3500 ms, for T₂-weighted SE sequences).

Figure 4.

(A) T₁-weighted MR images and T₁-mapping pseudo-color images of popliteal lymph nodes before and after injection subcutaneously of MnC-10, MnC-20, MnC-50 and MnC-127 at the dosage of 1000 μg/kg BW, respectively (scalar bar = 1 mm); quantitative analysis of the signal intensity from T₁-weighted MR images (B) and T₁ relaxation time from T₁-mapping images (C). *P < 0.05; **P < 0.01.

Figure 5.

(A) T₁-weighted MR images and T₁-mapping pseudo-color images of popliteal lymph nodes before and after injection subcutaneously of MnC-20 at the dosage of 750, 500, 250 and 125 μg/kg BW, respectively (scalar bar = 1 mm); quantitative analysis of the signal intensity from T₁-weighted MR images (B) and T₁ relaxation time from T₁-mapping images (C), and the data for 1000 μg Mn/kg BW were reproduced from Fig. 4. *P < 0.05; **P < 0.01.

Figure 6.

(A) Representative T₁-weighted MR images, T₁-weighted pseudo-color images and T₁-mapping pseudo-color images of normal popliteal lymph nodes (L) and metastatic sentinel lymph nodes (R) before and after injection subcutaneously of MnC-20 at the dosage of 125 μg/kg BW (scalar bar = 1 mm); quantitative analysis of the signal intensity from T₁-weighted MR images (B) and T₁ relaxation time from T₁-mapping images (C) (n = 4, *P < 0.05; **P < 0.01). (D) H&E staining images of normal popliteal lymph nodes and metastatic sentinel lymph nodes: area inside the red line contains tumor cells.

Scheme 1.

Schematic illustration of C18-PhDTA and polyethylene glycol monooctadecyl ether (C18En) assembled into mixed micelles, and further chelated with manganese to form manganese-based hybrid micellar complexes.