(⁹⁹m)Tc-bisphosphonate-iron oxide nanoparticle conjugates for dual-modality biomedical imaging

Abstract

The combination of radionuclide-based imaging modalities such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) with magnetic resonance imaging (MRI) is likely to become the next generation of clinical scanners. Hence, there is a growing interest in the development of SPECT- and PET-MRI agents. To this end, we report a new class of dual-modality imaging agents based on the conjugation of radiolabeled bisphosphonates (BP) directly to the surface of superparamagnetic iron oxide (SPIO) nanoparticles. We demonstrate the high potential of BP-iron oxide conjugation using (⁹⁹m)Tc-dipicolylamine(DPA)-alendronate, a BP-SPECT agent, and Endorem/Feridex, a liver MRI contrast agent based on SPIO. The labeling of SPIOs with (⁹⁹m)Tc-DPA-alendronate can be performed in one step at room temperature if the SPIO is not coated with an organic polymer. Heating is needed if the nanoparticles are coated, as long as the coating is weakly bound as in the case of dextran in Endorem. The size of the radiolabeled Endorem (⁹⁹m)Tc-DPA-ale-Endorem) was characterized by TEM (5 nm, Fe₃O₄ core) and DLS (106 ± 60 nm, Fe₃O₄ core + dextran). EDX, Dittmer-Lester, and radiolabeling studies demonstrate that the BP is bound to the nanoparticles and that it binds to the Fe₃O₄ cores of Endorem, and not its dextran coating. The bimodal imaging capabilities and excellent stability of these nanoparticles were confirmed using MRI and nanoSPECT-CT imaging, showing that (⁹⁹m)Tc and Endorem co-localize in the liver and spleen In Vivo, as expected for particles of the composition and size of (⁹⁹m)Tc-DPA-ale-Endorem. To the best of our knowledge, this is the first example of radiolabeling SPIOs with BP conjugates and the first example of radiolabeling SPIO nanoparticles directly onto the surface of the iron oxide core, and not its coating. This work lays down the basis for a new generation of SPECT/PET-MR imaging agents in which the BP group could be used to attach functionality to provide targeting, stealth/stability, and radionuclides to Fe₃O₄ nanoparticles using very simple methodology readily amenable to GMP.

Figure 1

General structure of a bisphosphonate (BP).

Figure 2

BP (DPA-ale) binding to iron oxide nanoparticles (Endorem): (A) Silica TLC spots of Endorem (left) and DPA-ale-Endorem (right) after Dittmer’s staining. Blue color demonstrates presence of phosphorus; (B, C) TEM pictures (top) and DLS studies (bottom) of Endorem (B) and DPA-ale-Endorem (C). (D, E) EDX studies of Endorem (D) and DPA-ale-Endorem (E). The inset is a magnification of the range between 0-5 keV. Note the presence of phosphorus in (E). A peak due to the presence of silicon is seen in (D) and is most likely to be due to contamination of the sample with vacuum grease.

Figure 3

In vitro imaging studies. (A) MRI relaxivity measurements of Endorem (closed circles, continuous line) and DPA-ale-Endorem (open triangles, discontinuous line) at 9.4 T. The slope of the linear fit represents the relaxivity of each sample (r₂). (B) T₂*-weighted MR phantom study of two samples containing the same amount of ^99mTc-DPA-ale-Endorem. In the left tube the nanoparticles were in solution, whereas in the right tube the particles had been pelleted by centrifuging. There is a strong negative signal from the pellet in the right tube. (C) NanoSPECT-CT image of the same sample from (B). There is a strong focal SPECT signal in the right sample, where ^99mTc-DPA-ale-Endorem had concentrated, whereas the signal in the left tube is diffuse.

Figure 4

Dual-modality in vivo studies. Short-axis view (top) and coronal view (bottom) images: (A) T₂*-weighted MR images before injection of ^99mTc-DPA-ale-Endorem, (B) T₂*-weighted MR image 15 min post-injection and (C) nanoSPECT-CT image of the same animal in a similar view 45 min post-injection. Contrast in the liver (L) and spleen (S) changes after injection due to accumulation of ^99mTc-DPA-ale-Endorem, in agreement with the nanoSPECT-CT image which shows almost exclusively liver and spleen accumulation of radioactivity. MR images were acquired with a TE of 2 ms.

Figure 5

In vivo studies. Whole body SPECT-CT maximum intensity projection (left) and biodistribution studies (right) of ^99mTc-DPA-ale-Endorem (A) and ^99mTc-DPA-ale (B). Biodistribution values represent the mean ± SD of the % ID/g (n = 3 mice).

Scheme 1

(A) Synthesis of ^99mTc-DPA-ale and (B) schematic representation of the synthesis of radiolabeled SPIO nanoparticles ^99mTc-DPA-ale-SPIO (top) and ^99mTc-DPA-ale-Endorem (bottom).