A GPC3-specific aptamer-mediated magnetic resonance probe for hepatocellular carcinoma

Abstract

Purpose: To construct and test a hepatocellular carcinoma (HCC)-targeted magnetic resonance probe based on a glypican-3 (GPC3)-specific aptamer (AP613-1) with ultrasmall superpara-magnetic iron oxide (USPIO).

Methods: Oleic acid-coated USPIO nanoparticles were modified with amino polyethylene glycol on the surface. Amino groups of the USPIO nanoparticles were reacted with the carboxyl group of 5' carboxyl-modified AP613-1, forming an aptamer-mediated USPIO (Apt-USPIO) probe. The material characterization of this probe including transmission electron microscopy (TEM), zeta potential, dynamic laser scattering, and magnetic behavior was carried out. The targeting efficiency and magnetic resonance imaging (MRI) performance of Apt-USPIO were evaluated both in vitro and in vivo with USPIO alone as a control. The cytotoxicity and bio-compatibility of Apt-USPIO and USPIO were analyzed by cell counting kit-8 tests in vitro and animal experiments in vivo.

Results: TEM imaging revealed that the Apt-USPIO nanoparticles were spherical in shape and well dispersed. Specific uptake of Apt-USPIO in Huh-7 cells could be observed using the Prussian blue staining test; however, no uptake of USPIO could be found. In vitro phantom T₂-weighted MRI showed a significant decrease of the signal intensity in Apt-USPIO-incubated Huh-7 cells compared to USPIO-incubated Huh-7 cells. In vivo T₂-weighted MRI showed significantly negative enhancement in the Huh-7 tumors enhanced with Apt-USPIO, whereas no enhancement was found with USPIO alone. Excellent biocompatibility of Apt-USPIO and USPIO was also demonstrated.

Conclusion: In this study, a molecular MRI probe which was highly specific to GPC3 on HCC was successfully prepared. Our results validated the targeted imaging effect of this Apt-USPIO probe in vivo for GPC3-expressing HCCs in xenograft mice.

Keywords: MRI; aptamer; carcinoma; glypican-3; hepatocellular; tumor-targeted imaging; ultrasmall superparamagnetic iron oxide.

Figure 1

Schematic diagram of the synthesis of AP613-1-mediated USPIO (Apt-USPIO). OA-coated USPIO nanoparticles are modified with DSPE-PEG₂₀₀₀-NH₂. Amino moieties (NH₂-) of USPIO nanoparticles and 5′ carboxyl (-COOH) of AP613-1 are reacted to construct the Apt-USPIO probe. Abbreviations: Apt-USPIO, aptamer-mediated USPIO; DSPE-PEG₂₀₀₀-NH₂, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS, N-hydroxysuccinimide; OA, oleic acid; USPIO, ultrasmall superparamagnetic iron oxide.

Figure 2

Characterization of Apt-USPIO and USPIO. (A and B) TEM images show that the Apt-USPIO nanoparticles disperse well, and are round in shape with an estimated magnetic core size of 10 nm. (C) The zeta potential is −17.9 mV for USPIO and −21.9 mV for Apt-USPIO. (D) The hydrodynamic diameter is 38.0 nm for USPIO nm and 45.2 nm for Apt-USPIO. Abbreviations: Apt-USPIO, aptamer-mediated USPIO; TEM, transmission electron microscopy; USPIO, ultrasmall superparamagnetic iron oxide.

Figure 3

Electrophoresis tests. The (A) nucleic acid image and the (B) corresponding color photograph display different positions of aptamer (AP613-1), Apt-USPIO, and USPIO bands, representing their different speeds of migration to the anode with agarose gel electrophoresis. Abbreviations: Apt-USPIO, aptamer-mediated USPIO; USPIO, ultrasmall super-paramagnetic iron oxide.

Figure 4

Immunofluorescence staining images of GPC3 show that Huh-7 cells are positively stained but L-02 cells are not. GPC3 represents the anti-GPC3 antibody binding (green) and DAPI locates the nuclei (blue). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.

Figure 5

Cytotoxicity of the USPIO and Apt-USPIO nanoparticles. In vitro viability study of Huh-7 cells in the presence of samples with varied concentrations for (A) 24 and (B) 48 h, and that of L-02 cells for (C) 24 and (D) 48 h. There is no significant reduction of cell viability with the increased concentration of USPIO or Apt-USPIO and the prolonged incubation time. Abbreviations: Apt-USPIO, aptamer-mediated USPIO; USPIO, ultrasmall superparamagnetic iron oxide.

Figure 6

Intracellular uptake experiments. Prussian blue staining images of the Huh-7 cells after 12 h of incubation with (A) USPIO and (B) Apt-USPIO display the blue granules in the cytoplasm of Apt-USPIO-incubated Huh-7 cells, but no stain was found in the USPIO-incubated control cells. No specific stain was found in the L-02 cells incubated with (C) USPIO and (D) Apt-USPIO. Abbreviations: Apt-USPIO, aptamer-mediated USPIO; USPIO, ultrasmall superparamagnetic iron oxide.

Figure 7

In vitro phantom MRI analysis. In vitro phantom T₂-weighted images of Huh-7 cells (A) and L-02 cells (B) incubated with various concentrations of Apt-USPIO and USPIO (0, 50, 100, 150, and 200 µg/mL) for 3 h, and curves for the corresponding signal intensities of (C) Huh-7 and (D) L-02 cells show that the signal decline in the Huh-7 cells incubated with Apt-USPIO is much more pronounced than the other groups. Abbreviations: Apt-USPIO, aptamer-mediated USPIO; MRI, magnetic resonance imaging; USPIO, ultrasmall superparamagnetic iron oxide.

Figure 8

In vivo MRI of xenograft tumors. (A) In vivo T₂-weighted MRI of the HCC xenograft models after injection of USPIO or Apt-USPIO. (B) T₂-weighted MR signal intensities of the Huh-7 tumors significantly decrease in the experimental (Apt-USPIO) group as compared to the control (USPIO) group. (C) RSE values in the experimental group are significantly higher than the control group at different time points. (D) Coronal and axial T₂-weighted MR images of tumor-bearing mice before and after intravenous injection show significant negative enhancement of the tumors (red circles) at different time points in the Apt-USPIO group. (E) There is no decline in the signal intensities of the tumors (yellow circles) in the corresponding USPIO group. Abbreviations: Apt-USPIO, aptamer-mediated USPIO; HCC, hepatocellular carcinoma; MRI, magnetic resonance imaging; RSE, relative signal enhancement; USPIO, ultrasmall superparamagnetic iron oxide.

Figure 9

In vivo biocompatibility analyses. (A) After intravenous administration of USPIO, Apt-USPIO, and PBS (control), no significant difference in the body weight is found among the three groups of Kunming mice during the 30 days feeding. (B) H&E staining of the vital organs including heart, liver, spleen, lung, and kidney from these mice in the three different groups shows normal findings. Abbreviations: Apt-USPIO, aptamer-mediated USPIO; H&E, hematoxylin and eosin; PBS, phosphate buffer solution; USPIO, ultrasmall superparamagnetic iron oxide.