| Background and Objective:Hepatocellular carcinoma (HCC) is one of the most commonest malignant tumors in the world, especially in China. The incidence of HCC ranks the fifth place in all maglinant tumors, and it is on rise year by year. The prognosis of HCC is very poor. The median survival time of HCC patients are only 8 months without active treatments. However, the prognosis would improve obviously, if the patients can be treated in early stage. Therefore, early diagnosis of HCC is vital to successful surgical treatment and is key to improve prognosis.In clinic, magnetic resonance image is common method to diagnose HCC, but there are atypical imaging finding in MRI. Especially in HCC patients who were progressed form liver cirrhosis, the finding are easy to be affected by focal nodule, resulting in decreased accuracy. And because common contrast agent is difficult for specific diagnosis, it is hard to distinguish HCC. In recent years, molecular imaging techniques, especially in MRI, develop rapidly. It can detect targeting lesion sensitively by using high-affinity molecular probe, and analyze lesion at molecular level. Hence molecular imaging is one kind of potential tools for HCC early diagnosis.The selection of target point and ligand concern the sensibility and specificity of diagnosis. Recent research has testified the overexpression of GPC3 protein in a large proportion of HCC,which is primarily linked to the cell surface with high specificity. Especially, GPC3 can be employed to identify early HCC. Studies in animals showed that GPC3-targeting monoclonal antibody mediated MRII could detect small (<10mm) HCC lesions. L5 peptide has the same targeting capability as monoclonal antibody besides its superiority to the latter, such as low molecular weight, cheap and non-immunogenicity.In order to improve efficiency of contrast enhancement, we apply biotin-avidin system (BAS). BAS possess high affinity and specificity, and is easy to conjugate ligands and agents without changing their function. Compared to a directly labeled MoAb approach, an antibody-guided pretargeting approach, through a stable bridge of biotin-avidin, holds promise in improving the tumor/nontumor ratio.In this paper, we plan to evaluate the value of pretargeting technology in vitro MR imaging of L5 peptide guided streptavidin-conjugated and polyethylene glycol modification protected ultra-small superparamagnetic iron oxide(SA-PEG-USPIO) to HCC via GPC3 receptor.Contents and Methods:1. Direct immunofluorescent method of HepG2 Cells with FAM-Labeled L5 PeptidesCells were cultured on six-chamber slides, and nonspecific bingding of peptides was blocked with 1% BSA for 60 min at 4℃ for 1 h. In experimental group, the slides were incubated with O.lmg/ml of FAM-labeled L5 or FAM in PBS/1% BSA, and then 4’,6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. In blocking group, the slides were incubated with lmg/ml L5, then washed and incubated with 0.1 mg/ml of FAM-labeled L5. Imaging was obtained from fluorescent microscope.2. Indirect immunofluorescence assay verify GPC3 expression on HepG2 cellsHepG2 cells were cultured on six-chamber slides. After fixed with 4% paraformaldehyde solution, the slides were incubated with anti-GPC3 first antibody (diluted 100 times with 5% bull serum albumin) in 4 ℃ for all night, then washed and incubated with FITC-conjugated second antibody. Imaging was obtained from fluorescent microscope.3. Preparation of SA-PEG-USPIOSA-PEG-USPIO were prepared by amide bond. PEG-USPIO was dissolved in 0.1 M MES buffer solution, then 2 mg EDC and 5.5mg sulfo-NHS were added and react for 15 min at room temperature.2-mercaptoethanol was used to quench EDC. The solvent was removed by centrifuging ultrafiltration, and the nanoparticles was resuspended in PBS buffer solution. Add 3 mg SA and react for 2 h with shaking at room temperature, and quench the reaction by 30 mM ethanolamine. Finally, the solution was centrifuging ultrafilted and adjust the concentration to 1 mg Fe/ml in PBS.4. CharacterizationThe surface charge and mean hydrodynamic size distribution of PEG-USPIO and SA-PEG-USPIO were determined in PBS using Malven Zeta 3000HS (Malvern Instruments, Malvern, UK) operating at 633.0 nm and 25.00±0.05 ℃.5. Magnetic property measurementsThe T2 relaxivity (r2) of PEG-USPIO and SA-PEG-USPIO was measured at 3.0 T MR system (GE, USA) using T2 mapping sequence (TR=2000 ms, TE=20,40,60, 80 ms, FOV=75×75 mm) in a knee coil. Both of them were prepared in 0.04,0.06, the Fe concentration (mM).6. Cytotoxicity assayIn vitro cytotoxicity of PEG-USPIO and SA-PEG-USPIO was evaluated in HL-7702 cells. At first, HL-7702 were seeded in 96-well plates with the density of 2×104 cells/well for 24 h, and then incubated with PEG-USPIO or SA-PEG-USPIO at different concentrations (0.4,0.8,1.2,1.6,2.0 and 2.4 mM Fe in DMEM) for 24 h. Before of the addition of dimethyl sulfoxide (150μL),20 μL of MTT (5 mg/mL) was added to each wells and incubated for 4h. The OD490 value of the reaction mixture on each well was measured by BIOTEK ELX800 microplate reader. And we calculated the cells viabilities as the percentage of live cells over control group only contained cells.7. Prussian blue staining of cellular labelHepG2 and HL-7702 cell were grown onto the 12-well plate with 1.5 × 105 cells/well for 24h. The supernatant was discard and the reagent was added to different wells by using different methods respectively. For L5-BT/SA-PEG-USPIO group,0.2 mg/ml of L5-BT was added as pretargeting for 1h, and cells were incubated with SA-PEG-USPIO (Fe concentration of 1.8 mM) for 2 h after washed three times with PBS. For the SA-PEG-USPIO group, cells were incubated with SA-PEG-USPIO at the Fe concentration of 1.8 mM for 2 h. The untreated cell was regarded as control group. Subsequently the cells was washed three times, and fixed with 4% paraformaldehyde solution. Then the cells were stained with potassium ferrocyanate solution for 20min at room temperature, rewashed, and counterstained with nuclear fast red.8. In vitro MRIHepG2 and HL-7702 cell were grouped according to the method of last step, and seeded on 100 mm diameter cell culture dishes. After incubation, cells were detached and resuspended in 1% agarose at a cell concentration of 1.5×105 cells/mL, and then transferred into 1.5 ml centrifuge tubes. Pseudocolor images of T2 value of the tubes were acquired using T2 mapping sequence (TR=2000 ms, TE=20,40,60,80 ms, FOV=75×75 mm) in a brain coil, and T2-weighted images were performed with SE sequence (TR=2500 ms, TE=96 ms, NEX=4, FOV=75×75 mm, thickness=2 mm, interval=2 mm). The T2WI signal intensity was normalized to that of 1% agarose.5 replicates for each group were performed.9. In vivo MRIHCC xenografts were treated with biotin-conjuncted L5 peptide and SA-PEG-USPIO probe in experimental group, PEG-USPIO probe in control group. Spin echo T2-weighted imaging was performed on 3.0 T MR scanner. Finally, MR images, immunohistochemistry and Prussian blue staining of HCC xenograft were used to evaluate the value of GPC3-targeted MR imaging.Results:1. In order to test the binding capacity of L5 peptide to HCC, immunofluorescence was performed by FAM. Fluorescent signal was found in the cytoplasm and surface of HepG2 cells, but not in HL-7702 cells. And only little FAM bound to the HepG2 cells. Blocking group was designed to show binding specificity of L5 for HCC, and there was decreased fluorescent signal in HepG2 cells. And immunofluorescence showed HepG2 cells express GPC3 receptor.2. Dynamic light scattering was used to measure hydrodynamic diameters, zeta potential and distribution of PEG-USPIO and SA-PEG-USPIO. PEG-USPIO and SA-PEG-USPIO had respective average hydrodynamic diameters of 22.73 nm and 35.97 nm with a polydispersity index of 0.207 and 0.169, and their Zeta potential were 4.22 mV and -7.91 mV.3. The T2 relaxivities of PEG-USPIO and SA-PEG-USPIO were measured before MRI experiment (Figure 4). The pseudocolor images of T2 value displayed the color of the tubes deepened with the increase of Fe concentration. The T2 relaxivity values of them were 0.1394×103 mM-’s-1 and 0.1039×103 mM-1s-1 respectively. We could find that after SA bingding, the T2 relaxivity values were still high enough for obtain high MRI sensitivity.4. To evaluate the cytototxicity of nanoparticles, HL-7702 were incubated with PEG-USPIO and SA-PEG-USPIO for 24 h by MTT assay. The cell viability had not obvious decrease in various concentrations range from 0.4 to 2.4 mM Fe. We could find that the cell viability of every concentrations remained above 80%. It indicated that both of nanoparticle are low-toxic for further assay.5. In order to evaluate the binding capacity of nanopartical in vitro, Prussian blue staining was performed. In L5-BT/SA-PEG-USPIO group, blue granules could be seen in most of the cells, them were located at cytoplasm and membrane. Only a little weak blue spots appear in other group.6. In order to validate the nanopartical MR imaging performance, we measured the normalized signal intensity and obtained the pseudocolor images of T2 value of HepG2 and HL-7702 cells in different groups. T2W MR images showed a dramatic signal drop in L5-BT/SA-PEG-USPIO group of HepG2, while the pseudocolor images of T2 value showed a significant deepened image. A quantitative measure showed the normalized MR signal intensity of this group was much lower than those of other group (L5-BT/SA-PEG-USPIO group of HL-7702, SA-PEG-USPIO group of HepG2 and HL-7702, P<0.05), and SNK method was used to multi-compare the four groups with each other.7. T2WI signal intensity of xenografts decreased to a minimum at 10 min in both group, and it began to rise slowly in pretargeting group, especially at 2 h,4 h and 6 h. However, the signal intensity returned to baseline after 1h in control group. There were blue-stained particles in and around the cancer cell after Prussian blue staining. Immunofluorescence showed HepG2 cells express GPC3 receptor in the same area. Relative signal intensity-time diagram can show a signal intensity plateau in pretargeting group. The signal intensities on T2WI at 1 h,2 h,4 h and 6 h in pretargeting group were significantly higher (P<0.05) than that in control group.Conclusions:By the pretargeting method, L5 peptide guided SA-PEG-USPIO has effective targeting ability to HCC. It will be useful for particular HCC diagnosis. |
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