Synthesis, Characterization, and Application of Core-Shell Co0.16Fe2.84O4@NaYF4(Yb, Er) and Fe3O4@NaYF4(Yb, Tm) Nanoparticle as Trimodal (MRI, PET/SPECT, and Optical) Imaging Agents

Abstract

Multimodal nanoparticulate materials are described, offering magnetic, radionuclide, and fluorescent imaging capabilities to exploit the complementary advantages of magnetic resonance imaging (MRI), positron emission tomography/single-photon emission commuted tomography (PET/SPECT), and optical imaging. They comprise Fe3O4@NaYF4 core/shell nanoparticles (NPs) with different cation dopants in the shell or core, including Co0.16Fe2.84O4@NaYF4(Yb, Er) and Fe3O4@NaYF4(Yb, Tm). These NPs are stabilized by bisphosphonate polyethylene glycol conjugates (BP-PEG), and then show a high transverse relaxivity (r2) up to 326 mM(-1) s(-1) at 3T, a high affinity to [(18)F]-fluoride or radiometal-bisphosphonate conjugates (e.g., (64)Cu and (99m)Tc), and fluorescent emissions from 500 to 800 nm under excitation at 980 nm. The biodistribution of intravenously administered particles determined by PET/MR imaging suggests that negatively charged Co0.16Fe2.84O4@NaYF4(Yb, Er)-BP-PEG (10K) NPs cleared from the blood pool more slowly than positively charged NPs Fe3O4@NaYF4(Yb, Tm)-BP-PEG (2K). Preliminary results in sentinel lymph node imaging in mice indicate the advantages of multimodal imaging.

Figure 1

TEM images at low and high magnifications, and the size distribution of Co_0.16Fe_2.84O₄@NaYF₄(Yb, Er) NPs (a–c) and Fe₃O₄@NaYF₄(Yb, Tm) NPs (d–f). The particle size was determined on TEM and N is the number of particles counted for size analysis.

Figure 2

HRTEM studies of NPs: (a) HRTEM images of Fe₃O₄@NaYF₄(Yb, Tm); (b) fast Fourier transform of the selected area in part a, showing two sets of diffraction patterns. The diffraction pattern marked in blue belonged to cubic Fe₃O₄, and the one marked in red was assigned as cubic NaYF₄; (c) high angle annular dark field image of Fe₃O₄@NaYF₄(Yb, Tm), showing the Z contrast difference between the shell and core of particles induced by a slightly higher average atomic number in the shell after doping with heavy atoms Yb and Tm; (d) HRTEM image revealed the core–shell structure of NP Co_0.16Fe_2.84O₄@NaYF₄(Yb, Er). Atomic lattice fringes 2.97 and 4.14 Å corresponded to (022) and (200) planes of Fe₃O₄, respectively. The inset is a fast Fourier transform of the micrograph.

Figure 3

(a) Curve of relaxivity against the concentration of Fe+Co for Co_0.16Fe_2.84O₄@NaYF₄(Yb, Er)-BP-PEG at 3T and 7T) (concentration of Fe and Co determined by ICP-MS); (b) up-conversion spectrum of Co_0.16Fe_2.84O₄@NaYF₄(Yb, Er)-BP-PEG under excitation by a 980 nm laser; (c) transmitted infrared image of HeLa cell culture in monolayer (gray scale) incubated with Co_0.16Fe_2.84O₄@ NaYF₄(Yb, Er)-BP-PEG and fluorescence emission image (green, 490–560 nm) overlaid. Excitation wavelength 980 nm. Arrows show green fluorescence from cell-incorporated particles.

Figure 4

PET/MR images showing the dynamic biodistribution of ¹⁸F radiolabeled NPs. MR images were taken immediately after PET scans, 2 h post the injection of NPs, and they were fused with PET images taken at three different time intervals (0–15 min, 45–60 min, and 105–120 min). (a) PET/MR fused image at 0–15 min, showing radioactivity mainly confined to blood pool with minor uptake in liver; (b) PET/MRI fused image at 45–60 min showing increased accumulation in liver and minor uptake in skeleton; and (c) PET/MR fused image at 105–120 min showing increased uptake in skeleton and bladder; (d) whole body PET image showing uptake of radiolabeled negatively charged NPs Co_0.16Fe_2.84O₄@NaYF₄(Yb, Er)-BP-PEG (10K) (maximum intensity projection, 30–45 min), showing radioactivity mainly confined to blood pool, with little activity in bone or bladder; (e) MR image of the mouse prior to the injection of Co_0.16Fe_2.84O₄@NaYF₄(Yb, Er)-BP-PEG (10K) NPs; (f) MR image of the mouse 2 h post injection of Co_0.16Fe_2.84O₄@NaYF₄(Yb, Er)-BP-PEG (10K) NPs. The series of images (a) to (d) shows predominantly blood pool retention of the labeled NPs at early stages post injection, giving way to liver uptake and then bone and bladder, consistent with release of radioactivity from nanoparticles in liver as free fluoride.

Figure 5

PET/MRI images of a normal young C57BL/6 mouse showing LNs with dual contrast provided by ¹⁸F-labeled Co_0.16Fe_2.84O₄@NaYF₄(Yb, Er)-BP-PEG: (a) schematic diagram showing the connections between lymph nodes and the injection point (mouse in supine position); (b) whole body PET image showing uptake of radiolabeled NPs 7 h post injection (maximum intensity projection, mice in prone position); (c) MR image with darkening contrast at popliteal LN (coronal section); (d) PET image showing radioactivity at popliteal, inguinal, lumbar aortic, and aorta LN area (coronal section); and (e) PET/MRI fused image showing popliteal, inguinal, lumbar aortic, and aorta LN (coronal section). Some bone uptake of radioactivity is observed in (b), (d), and (e) due to gradual release of fluoride from the particles due to the long delay (7 h post injection of ¹⁸F radiolabeled NPs) between injection and imaging.

Figure 6

Lymph node PET/MRI imaging of a mouse with inflamed right leg using ¹⁸F-labeled Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG NPs (a–d) or with [¹⁸F]-fluoride only (e–g): (a) whole body PET image showing uptake of radiolabeled NPs (maximum intensity projection; bone uptake was observed due to gradual release of fluoride from NPs due to the 7 h delay post injection of NPs); (b) PET image showing popliteal and iliac lymph nodes (coronal section); (c) PET/MRI fused image (coronal section); (d) MR image (coronal section) with darkening contrast inside popliteal lymph node at left-rear (white circle) and “outside” lymph node at the inflamed right-rear (red circle) induced by injection of 30 μL 0.67 mg/mL lipopolyscchrade (LPS) 18 h prior to imaging, and at iliac lymph node; (e) PET image following injection of [¹⁸F]-fluoride showing no contrast in lymph nodes in the absence of NPs and prominent uptake by skeleton; (f) PET/MRI fused image following injection of [¹⁸F]-fluoride, showing no radioactivity associated with lymph nodes; (g) MR image showing no difference between normal popliteal lymph node at left-rear leg (white circle) and the inflamed lymph node at right-rear leg induced by injection of 30 μL 0.67 mg/mL LPS 18 h prior to imaging; and (h–k) enlarged MR images of corresponding lymph nodes.