| Glypican-3(GPC3)is a member of the glypican family.of heparin sulfateproteoglycans(HSPGs).This protein was reported to be highly expressed in hepatocellular carcinoma(HCC),but not in normal liver tissue,cirrhosis tissue or paracancerous tissue.L5(sequence:RLNVGGTYFLTTRQ)is a specific target peptide of GPC3 screened using bacteriophage display and transfection technique by Lee et al.Previously,we had developed L5 to be a GPC3 targeted PET tracerbylabeling it with positron emitter of 18F and successfully visualized the GPC3 positive HCC tumors in vivo using microPET/CT.However,18F-labeled L5 showed a low tumor/liver ratio because high accumulation of radioactivity in liver due to the hepatobiliary excretion,which will hamper the detection of HCC.In order to decrease hepatobiliary excretion,in present study,we added a hydrophilic group of GGGRDN(L)to the NH2 end of L5 to obtain a modified peptide,namedL-L5,and then conjugated L-L5 with NOTA for 18F-AlF chelated radiolabeling.It is expected that the introduction of the hydrophilic group to the L5 pepetide will help to inscrease the water-solubility of probe and reduce the background radioactivity in the liver,which may improve the detectability of PET/CT for the HCC tumor.Purposes1.To develop a novel hydrophilic GPC3 targeted PET probe(18F-AlF-NOTA-L-L5)by conjugating a hydrophilic group of GGGRDN(L)to L5 peptide and radiolabeling it with 18F-AlF using a chelate chemistry.2.To investigate whether the introduction of hydrophilic group can reduce hepatobiliary excretion of novel probe and improve the tumor/liver ratio,comparing to intact 18F-AlF-NOTA-L5.Method1.GPC3-targeting peptides of L5 and L-L5 were synthesized using the method of solid phase chemical synthesis.L5 and L-L5 were then conjugated with NOTA to obtain the modified NOTA-L5 and NOTA-L-L5.All the peptides were isolated and purified by high performance liquid chromatography(HPLC),and identified by mass spectrum(MS).2.The affinity of NOTA-L5 and NOTA-L-L5 to GPC3 protein was determined using surface plasmon resonance(SPR)measurements.3.The radiolabeled probes 18F-AlF-NOTA-L5 and 18F-AlF-NOTA-L-L5 were produced using 1-pot chelated chemistry.Their labeling yields and radiochemical purities were then determined using radio-HPLC.4.The Octanol/Water partition coefficients of the above PET probes were performed using a gamma counter.In vitro stability of the above PET probeswas measured using radio-HPLC,and the in vivo stability was measured using a gamma counter.5.Cell uptake and efflux studies were used to assess the binding capability and washout of 18F-AlF-NOTA-L5 and 18F-AlF-NOTA-L-L5 with GPC3 positive HepG2 cells and GPC3 negative RH7777 cells.6.The GPC3 positive HepG2 tumor models and GPC3 negative RH777 tumor models were established.MicroPET/CT was performed to evaluate the tumor detective capability and the bio-radioactivity distribution of 18F-AlF-NOTA-L5 and 18F-AlF-NOTA-L-L5 in vivo.7.Immunohistochemical staining was performed to determine the GPC3 expression in the HepG2 and RH7777 tumor tissues.Results1.The peptides of L5,L-L5,NOTA-L5 and NOTA-L-L5 were successfully synthesized.Mass chromatographic analysis(ESI)showed the molecular mass(MS)(m/z:Da)of produced L5,L-L5,NOTA-L5 and NOTA-L-L5 were 1626.3,2183.1,2076.3 and 2632.8,respectively,which were in accordance with their calculated MS of 1625.86,2182.40,2076.37 and 2632.91.After purified by preparative HPLC,all the purities of L5,L-L5,NOTA-L5 and NOTA-L-L5 were determined and were greater than 95.0%.2.The surface plasmon resonance(SPR)assay showed the dissociation constants(KD)of NOTA-L5 and NOTA-L-L5 to GPC3 were 1.01 × 10-7 mol and 6.33 × 10-8mol,respectively.3.18F-AlF-NOTA-L5 and 18F-AlF-NOTA-L-L5 were successfully radiolabeled using the 18F-AlF chelate chemistry with the yields of 79.98±8.04%and 54.81±9.05%,respectively.After purified by Sep.Pek C18,the radiochemical purifies were measured to be 97.17±1.03%and 100±0%,respectively.4.Octanol/Water partition coefficient(LogP)of 8F-AlF-NOTA-L5 and 18F-AlF-NOTA-L-L5 were-2.10 ±0.07 and-2.42±0.09,respectively and their difference was significant(t = 5.482,P = 0.002).5.All 18F-AlF-NOTA-L5 and 18F-AlF-NOTA-L-L5 were stable in vitro.More than 94%intact radioactivity of 18F-AlF-NOTA-L5 and 98%intact radioactivity of 18F-AlF-NOTA-L5 were found in PBS and serum after 1h incubation at 37 ?.In vivo,intact radioactivity of 18F-AlF-NOTA-L5 was found 97.69±2.51%in the HepG2 tumor,96.06±0.54%in the liver,96.68±1.55%in the blood,but only 54.94±2.12%in the kidneys and 3.31 ±0.20%in the the urine,respectively.Similarly,intact radioactivity of 18F-AlF-NOTA-L5 was found 93.01±2.98%in the HepG2 tumor,92.95±2.77%in the liver,93.57±1.38%in the blood,but only 7.70±2.56%in the kidneys and 1.00±0.01%in the urine,respectively.6.Immunohistochemical staining demonstated that GPC3 was highly expressed in HepG2 tumor,butnegatively expressed in RH7777 cells.7.In vitro cellular uptake assays revealed that the uptake of 18F-AlF-NOTA-L-L5 was similar to that of 18F-AlF-NOTA-L5 by GPC3 positive HepG2 cells.However,the uptake of 18F-AlF-NOTA-L-L5 by HepG2 cells was significantly higher than that of GPC3-negative RH7777 cells.Two PET tracers all reached the plateau at 60min.The washout of 18F-AlF-NOTA-L-L5 was slightly faster than that of 18F-AlF-NOTA-L5.9.MicroPET/CT showed the HepG2 tumors were clearly visualized by 18F-AlF-NOTA-L-L5 and 18F-AlF-NOTA-L5 at 30min,60min and 120 min after intravenously injection.In vivo radioactivity biodistribution demonstrated that the uptake of 18F-AlF-NOTA-L-L5 in tumor at 30 and 60min after injection weresignificantly higher than those of 18F-AlF-NOTA-L5(30min:3.10 ± 0.26%ID/g vs 2.28 ± 0.33%ID/g,t = 3.935,P = 0.008;60min:3.27 ± 0.34%ID/g vs 1.90 ±0.65%ID/g,t=3.757,P=0.016,respectively).The uptake of 18F-AlF-NOTA-L-L5 in the liver were significant lower than that of 18F-AlF-NOTA-L5 at 30min and 60min,but not at 120min(30min:6.35±2.54%ID/g vsl.93±0.39%ID/g,t=3.449,P=0.038;60min:4.32±1.49%ID/g vs 1.85±0.37%ID/g,t=3.232,P=0.041;120min:2.30±0.21%ID/g vs 1.23±0.43%ID/g,t=2.118,P=0.079,respectively).The kidney uptake of 18F-AlF-NOTA-L-L5 was significantly higher than those of 18F-AlF-NOTA-L5(30min:37.55±3.79%ID/gvs12.83 ± 1.65%ID/g,t =6.426,P =0.001;60min:35.9±2.56%ID/gvs8.70±1.70%ID/g,t =11.798,P = 0.000;120min:31.86±3.8%ID/gvs2.57±0.17%ID/g,t=4.224,P?0.006).The tumor/non-tumor ratio of 18F-AlF-NOTA-L-L5 and 18F-AlF-NOTA-L5 in various organs were calculated.The tumor/liver uptake ratio of 18F-AlF-NOTA-L-L5.was higher than those of 18F-AlF-NOTA-L5 at 30min,60min and 120min after injection(30min:1.65±0.28vs0.39±0.11,t =8.314,P = 0.000;60min:1.82±0.38vs0.48±0.23,t =6.068,P=0.001;120min:1.17±0.31vs0.49±0.39,t =2.740,P = 0.034).18F-AlF-NOTA-L-L5 had also significantly higher tumor/intestine ratio than those of 18F-AlF-NOTA-L5(30min:2.70 ± 1.19 vs.0,64±0.35,t=3.324,P =0.016;60 min:2.74 ± 0.44 vs.0.71 ± 0.53,t = 5.863,P =0.001).However,18F-AlF-NOTA-L-L5 was found to have significantly lower tumor/kidney ratios than those of 18F-AlF-NOTA-L5(60 min:0.10±0.01 vs.0.26 ±0.11,t = 2.713,P = 0.035).Conclusion1.The 18F-AlF-NOTA-L5 and 18F-AlF-NOTA-L-L5 were successfully prepared by A118F chelation.The radiolabeling is simple,time-saving,and can be completed by the "one-pot method".It can also be carried out by modular automated production,which will be easily transfered to clinic.2.The introduction of the hydrophilic group GGGRDN to L5 peptide increases the hydrophilicity,however,the modify dose not affect the binding ability to GPC3 protein.3.18F-AlF-NOTA-L-L5 have a lowerradiolabeling yields than 18F-AlF-NOTA-L5.4.The stability of 18F-AlF-NOTA-L5 and 18F-AlF-NOTA-L-L5 were high in vitro and in vivo.5.Cell uptake of 18F-AlF-NOTA-L-L5 is similar to that of 18F-AlF-NOTA-L5,but washout is slightly higher than 18F-AlF-NOTA-L5.The cell uptake of 18F-AlF-NOTA-L-L5 was GPC3 specificity with high uptake in GPC3 positive HepG2 cells,but low in GPC3 negative RH777 cells.6.18F-AlF-NOTA-L-L5 microPET/CT can visualize the GPC3 positive tumor(HepG2 tumor)clearly in vivo,liking 18F-AlF-NOTA-L-L5.However,by introduction the hydrophilic linker,18F-AlF-NOTA-L-L5 showed high urinary excretion,but low hepatobiliary excretion,which was significantly different to 18F-AlF-NOTA-L-L5.As a result,the radioactivity background in the liver decrease significantly and the tumor/liver and tumor/intestine weresccordingly improved. |
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