Published online Jun 28, 2019. doi: 10.3748/wjg.v25.i24.3030
Peer-review started: February 25, 2019
First decision: March 20, 2019
Revised: April 3, 2019
Accepted: May 18, 2019
Article in press: May 18, 2019
Published online: June 28, 2019
Hepatocellular carcinoma (HCC) ranks second in terms of cancer mortality worldwide. Molecular magnetic resonance imaging (MRI) targeting HCC biomarkers such as alpha-fetoprotein (AFP) or glypican-3 (GPC3) offers new strategies to enhance specificity and help early diagnosis of HCC. However, the existing iron oxide nanoparticle-based MR molecular probes singly target AFP or GPC3, which may hinder their efficiency to detect heterogeneous micro malignant HCC tumors < 1 cm (MHCC). We hypothesized that the strategy of double antibody-conjugated iron oxide nanoparticles which simultaneously target AFP and GPC3 antigens may potentially be used to overcome the tumor heterogeneity and enhance the detection rate for MRI-based MHCC diagnosis.
To synthesize an AFP/GPC3 double antibody-labeled iron oxide MRI molecular probe and to assess its impact on MRI specificity and sensitivity at the cellular level.
A double antigen-targeted MRI probe for MHCC anti-AFP–USPIO–anti-GPC3 (UAG) was developed by simultaneously conjugating AFP andGPC3 antibodies to a 5 nm ultra-small superparamagnetic iron oxide nanoparticle (USPIO). At the same time, the singly labeled probes of anti-AFP–USPIO (UA) and anti-GPC3–USPIO (UG) and non-targeted USPIO (U) were also prepared for comparison. The physical characterization including morphology (transmission electron microscopy), hydrodynamic size, and zeta potential (dynamic light scattering) was conducted for each of the probes. The antigen targeting and MRI ability for these four kinds of USPIO probes were studied in the GPC3-expressing murine hepatoma cell line Hepa1-6/GPC3. First, AFP and GPC3 antigen expression in Hepa1-6/GPC3 cells was confirmed by flow cytometry and immunocytochemistry. Then, the cellular uptake of USPIO probes was investigated by Prussian blue staining assay and in vitro MRI (T2-weighted and T2-map) with a 3.0 Tesla clinical MR scanner.
Our data showed that the double antibody-conjugated probe UAG had the best specificity in targeting Hepa1-6/GPC3 cells expressing AFP and GPC3 antigens compared with single antibody-conjugated and unconjugated USPIO probes. The iron Prussian blue staining and quantitative T2-map MRI analysis showed that, compared with UA, UG, and U, the uptake of double antigen-targeted UAG probe demonstrated a 23.3% (vs UA), 15.4% (vs UG), and 57.3% (vs U) increased Prussian stained cell percentage and a 14.93% (vs UA), 9.38% (vs UG), and 15.3% (vs U) reduction of T2 relaxation time, respectively. Such bi-specific probe might have the potential to overcome tumor heterogeneity. Meanwhile, the coupling of two antibodies did not influence the magnetic performance of USPIO, and the relatively small hydrodynamic size (59.60 ± 1.87 nm) of double antibody-conjugated USPIO probe makes it a viable candidate for use in MHCC MRI in vivo, as they are slowly phagocytosed by macrophages.
The bi-specific probe presents enhanced targeting efficiency and MRI sensitivity to HCC cells than singly- or non-targeted USPIO, paving the way for in vivo translation to further evaluate its clinical potential.
Core tip: The single targeting of existing hepatocellular carcinoma-targeted magnetic resonance imaging (HCC-targeted MRI) probes may weaken the detection efficiency due to biomarker associated tumor heterogeneity. Here, double antibody-conjugated ultra-small superparamagnetic iron nanoparticles (USPIO) were synthesized to simultaneously target HCC markers of alpha- fetoprotein (AFP) and glypican-3 (GPC3) antigens in Hepa1-6/GPC3 cells. Such probe showed higher cancer cell labeling efficiency than singly- or non-targeted USPIO probes by Prussian blue staining and in vitro MRI, indicating enhanced specificity and sensitivity of MRI diagnosis for micro hepatocellular carcinoma (MHCC). Meanwhile, USPIO with a small core (~5 nm) and hydrodynamic size (~60 nm) after antibody labelling may undergo slow phagocytosis, which could enhance liver tumor MRI contrast in the animal or clinical trial study.