Convenient and effective ICGylation of magnetic nanoparticles for biomedical applications

Abstract

Nanoprobes used for biomedical applications usually require surface modifications with amphiphilic surfactants or inorganic coating materials to enhance their biocompatibility. We proposed a facile synthetic approach for the phase transfer of hydrophobic magnetic nanoparticles by the direct adherence of fluorescent probes, without any chemical modifications, for use as a magnetic resonance (MR)/near-infrared (NIR) fluorescence bimodal imaging contrast agent. Indocyanine green (ICG) was used not only as an optical component for NIR imaging, but also as a surfactant for phase transfer with no superfluous moiety: we therefore called the process "ICGylation". Cell labeling and tracking in vivo with ICGylated magnetic nanoparticles were successfully performed by MR/NIR dual-mode imaging for three days, which showed remarkable biostability without any additional surface functionalization. We expect that this novel MR/NIR contrast agent demonstrating sensitive detection and simultaneous imaging capability can be used in diverse fields, such as the imaging and tracking of immune cells to confirm immunotherapeutic efficacy. The approach used could also be applied to other kinds of nanoparticles, and it would promote the development of advanced functional multimodal nanobioprobes.

Figure 1

Schematic illustration of preparation of ICGylated MNPs. Hydrophobic magnetic nanoparticles (MNPs) were coated with indocyanine green (ICG) molecules, which enable their phase transfer to aqueous media.

Figure 2

Characterization of ICGylated MNPs. (a) Transmission electron microscopy (TEM) image of MNPs in an organic solvent. (b) Magnetic separation procedure of ICGylated MNPs. (c) TEM image of ICGylated MNPs in water. (d) Size distribution of MNPs in an organic solvent and ICGylated MNPs in aqueous solvents. (e) Absorption and (f) fluorescence (emission scan at λ_ex = 765 nm) spectra of ICG and ICGylated MNPs. (g) FT-IR spectra of MNPs, ICG, and ICGylated MNPs (^*aromatic C = C stretches, ^**C-H out of plane bending). (h) T₂ relaxation rate (bottom, 1/T ₂, s⁻¹) and T₂-weighted MR images (top) as a function of Fe concentration for ICGylated MNPs. (i) Dark-field (left) and fluorescence microscopy (right) images of ICGylated MNPs at a single-particle level. MNPs are shown in the dark-field image; ICG is shown in the fluorescence image.

Figure 3

Enhanced photostability of ICG after ICGylation. (a) Dark-field (red), fluorescence (green), and merged images of ICGylated MNPs. MNPs are shown in the dark-field image; ICG is shown in the fluorescence image. In the merged image, the ICGylated MNPs appear yellow, whereas the free ICG molecules appear green. Yellow boxes (1–5) and green boxes (6–10) were selected as ROIs to measure the fluorescence signals for coated ICG and free (uncoated) ICG, respectively. (b) Averaged fluorescence intensities under continuous radiation (at 785 nm) for each ROI. Individual fluorescence intensities for each ROI are indicated in Supplementary Fig. 2.

Figure 4

In vitro MR/FL imaging of cells labeled with ICGylated MNPs. (a) In vitro microscope images and (b) flow cytometric analysis of the labeled DCs with ICGylated MNPs (top) and those of the unlabeled ones (bottom). Blue and red colors represent DAPI-stained nuclei and ICG fluorescence. The scale bar indicates 15 μm in (a). (c) Prussian blue staining of the labeled DCs incubated with 25 µg Fe/ml of ICGylated MNPs for 24 h. (d) T₂-weighted MR images (top) and T ₂ values (bottom) for the labeled DCs (5 × 10⁶) as a function of treated Fe concentration. (e) Same as (d) but as a function of the cell number with DCs labeled at a concentration of 100 µg Fe/ml of ICGylated MNPs.

Figure 5

In vivo MR/FL imaging of cells labeled with ICGylated MNPs. (a) In vivo T₂-weighted MR images of mouse popliteal lymph nodes at different times after injection of DCs labeled with ICGylated MNPs (L, left) and unlabeled (R, right) DCs (2 × 10⁶ cells). (b) Signal-to-noise ratios obtained from MR images of (a). (c) In vivo NIR fluorescence images of mouse popliteal lymph nodes after injection of the labeled DCs. (d) Contrast-to-noise ratio (CNR) values of the lymph nodes obtained from (c). ^***p < 0.001.