Theranostic imaging of liver cancer using targeted optical/MRI dual-modal probes

Abstract

The accurate preoperative detection and intraoperative navigation afforded by imaging techniques have had significant impact on the success of liver cancer surgeries. However, it is difficult to achieve satisfactory performance in both diagnosis and surgical treatment processes using any single modality imaging method. Here, we report the synthesis and characteristics of a novel dual-modality magnetic resonance imaging (MRI) and near-infrared fluorescence (NIRF) probe and verify its feasibility in nude mouse models with liver cancer. The probes are comprised of superparamagnetic iron oxide (SPIO) nanoparticles coated with liposomes to which a tumor-targeted agent, Arg-Gly-Asp peptides (RGD), and a NIRF dye (indocyanine green, ICG) have been conjugated. Specific targeting, biodistribution, and the imaging ability of the probes for MRI-NIRF were examined. Furthermore, we applied the dual-modality methodology toward the preoperative diagnosis and intraoperative guidance of radical resection in mouse models with both orthotopic liver tumors and intrahepatic tumor metastasis. The study demonstrated that both MRI and fluorescent images showed clear tumor delineation after probe injection (SPIO@Liposome-ICG-RGD). The contrast-to-noise ratio obtained from MRI was 31.9 ± 25.4 at post-injection for the preoperative diagnosis, which is helpful for detecting small tumors (0.9 ± 0.5 mm). The maximum tumor to background ratio of NIRF imaging was 2.5 ± 0.3 at 72 h post-injection for effectively capturing miniscule tumor lesions (0.6 ± 0.3 mm) intraoperatively. The novel MRI-NIRF dual modality probes are promising for the achievement of more accurate liver tumor detection and resection.

Keywords: MRI/optical; dual-modality; intraoperative navigation; liver cancer; preoperative diagnosis.

Figure 1. MRI and NIRF signal evaluation for SPIO@Liposome-ICG-RGD

A. The concentration of the probe was successively diluted from the left to right wells. For MRI, the negative contrast effect is clearly demonstrated with the decrease of SPIO concentration. However, for NIRF imaging, the optical signal decreases with diminishing ICG concentration. B. A linear correlation is observed between the fluorescence intensity and the probe concentration, whereas C. an inverse linear correlation can be seen between the T2-weighted MRI signal and the probe concentration. The R2 of the probe is 363.4 mM⁻¹ s⁻¹.

Figure 2

In vivo T2-weighted MR imaging before and after the injection of SPIO@Liposome-ICG-RGD A. or SPIO@Liposome-ICG B.

Figure 3. In vivo continuous observations of the orthotopic liver cancer model administrated with SPIO@Liposome-ICG-RGD

The contrast-to-noise ratio (CNR) of SPIO@Liposome-ICG-RGD was calculated as CNR = (SI_liver−SI_tumor)/SI_noise.

Figure 4. In vivo comparison of the biodistribution of SPIO@Liposome-ICG-RGD and SPIO@Liposome-ICG

A, B. In vivo continuous observations (120 h) of liver cancer xenografts administrated with SPIO@Liposome-ICG-RGD (A) or SPIO@Liposome-ICG (B) using FMI. Black circles indicate the regions of interest for calculating the tumor to background ratio (TBR) in each time point. The quantification of fluorescence intensity at the tumor sites reveals a higher accumulation of SPIO@Liposome-ICG-RGD at all observation points C. Comparison of TBR profiles of the two probes. The peak and maximum difference both occurred at 72 h post-injection, suggesting the optimal surgical window time D. Experiments were run in triplicate.

Figure 5. Theranostic imaging in the orthotopic liver tumors models

The MRI image before SPIO@Liposome-ICG-RGDs injection A. The MRI signal is obviously decreased in normal liver tissue (CNR: 34.2 ± 9.1) after targeted probe injection B. Surgical guidance by intraoperative FMI-NIR (fluorescence molecular imaging system) C. The implanted liver tumor tissue (blue arrow) exhibits obvious contrast (TBR: 2.4 ± 0.3) in color and texture with normal liver tissues D. The merge image of color and fluorescence demonstrates the excellent contrast E. The residual tumor node (blue arrow) after the first operation F. The residual tumor node exhibits obvious contrast (TBR: 2.5 ± 0.3) in color and texture with normal liver tissues G. The merged color and fluorescence image demonstrates the excellent contrast in the residual tumor node H. Identification of the residual tumor (0.6 ± 0.3 mm) after the initial resection I. Prussian blue staining confirmation of the targeting ability of SPIO@Liposome-ICG-RGDs J. HE staining confirmation of the liver tumor tissue K. Experiments were run in triplicate.

Figure 6. Theranostic imaging in the orthotopic liver tumors with intrahepatic metastasis

The MRI image before SPIO@Liposome-ICG-RGDs injection A. The MRI signal is obviously decreased in normal liver tissue (CNR: 14.6 ± 9.9) after targeting probe injection and the disseminated tumor nodes (0.9 ± 0.5mm) (blue arrow) can be clearly defined B. The presence of liver tumors as confirmed by bioluminescence imaging C. Surgical guidance by intraoperative FMI-NIR D. The implanted liver tumor tissue (0.7 ± 0.3 mm) (blue arrow) exhibits obvious contrast (TBR: 2.3 ± 0.5) in color and texture with normal liver tissues E. The merged color and fluorescence image demonstrates the excellent contrast F. Experiments were run in triplicate.

Figure 7. Schematic diagram for the synthesis of SPIO@Liposome-ICG-RGD

A. SPIO nanoparticles was coated with liposome (SPIO@Liposome). B. ICG molecules were loaded into the lipid layer of magnetic liposomes (SPIO@Liposome-ICG). C. RGDs were conjugated to obtain the SPIO@Liposome-ICG-RGD probes.