In vitro studies of glypican-3 targeting pretargeting technology for molecular MRI of hepatocellular carcinoma

Abstract

Objective To explore the value of pretargeting technology in vitro MRI of L5 peptide guided streptavidin-conjugated and polyethylene glycol modification protected ultra-small superparamagnetic iron oxide (SA-PEG-USPIO) to hepatocellular carcinoma (HCC) via glypican-3 (GPC3) receptor. Methods Direct immumofluorescence assay with carboxyfluorescein (FAM) labeled L5 and competitive inhibition was performed in HepG2 and HL-7702 cells. Imaging was obtained from fluorescent microscope. Immunoassay fluorescence images were carried out to determine the expression of GPC3 in HepG2 cell. PEG-USPIO conjugated with streptavidin was made by carbodiimide reaction, and the hydrodynamic diameters, Zeta potential and magnetic relaxivity of SA-PEG-USPIO and PEG-USPIO were measured. HL7702 cells were used for evaluate cells viability of SA-PEG-USPIO and PEG-USPIO. HepG2 and HL-7702 cells were used as experimental and control group respectively. Each of the two cell lines were further divided into three groups: L5-BT united SA-PEG-USPIO group, SA-PEG-USPIO group and control group. Prussian blue staining and MRI was preformed to observe the targeting efficacy of SA-PEG-USPIO respectively, and normalized T2 signal was recorded. The significant changes of normalized T2 signal intensity among groups was determine by using One-way analysis of variance. Results There were much more fluorescences on the membrane and cytoplasm of HepG2 cells than those on HL-7702 cells and cells of competition group. And indirect immunofluorescence images show the obvious expression of GPC3 in HepG2 cell. The SA-PEG-USPIO and PEG-USPIO nanoparticles had hydrodynamic diameters of (22.73 ± 3.31) and (35.97±5.19) nm, Zeta potential of them were (4.22±0.53) and (-7.91±1.22) mV and magnetic relaxivity were 0.139 4×103 and 0.103 9×103 mM-1s-1. Although the highest concentration of SA-PEG-USPIO and PEG-USPIO was 2.4 mmol/L, cells viability was greater than 80%. The most iron particle was observed in L5-BT united SA-PEG-USPIO group of HepG2 cells. In vitro MR, the normalized T2 signal intensity of HepG2 cells in L5-BT united SA-PEG-USPIO group, SA-PEG-USPIO group and control group were 39±7, 77 ± 12 and 93 ± 4. There was significant difference among those three groups (F=23.96,P 0.05). Conclusion By the pretargeting method, L5 peptide guided SA-PEG-USPIO has effective targeting ability to HepG2 cells in vitro. Key words: Carcinoma, hepatocellular; Magnetic resonance imaging; Molecular probes