Developing The Novel Molecular Probes Targeting To LAPTM4B Receptor And Evaluating Its Efficacy By The Fluorescent And PET Imaging Study In The Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is one of the most common cancers in the world,which ranks the third in cancer-induced death and second in domestic cancer mortality.Although many modalities of therapies have been applied to treat HCC,such as surgery,interventional therapy,radiotherapy and chemotherapy etc,they all have some limitations,especially for treating the advanced HCC.In recent years,targeted therapy has developed fast and shows its potential for treating the HCC.In order to obtain a successfully targeted therapy,the physician needs to determine whether the tumor has the high expression of the target.Developing a targeting imaging will be able to solve this dilemma,which can help the physician visualize the expression of a specific target in vivo clearly and comprehensively,and lead to a correct treatment decision.In 2000,an important receptor named transmembrane protein lysosomal transmembrane 4 beta,was discovered by Liu Junjian et al.This receptor is encoded by LAPTM4B gene located on chromosome 8q22.1 and was found to be highly expressed on the HCC,fetal liver tissue,but not on the paired non-cancerous liver tissue and normal liver tissue.This transmembrane protein had a potential to become a new treatment target.The polypeptide AP2H(amino acid sequence:IHGHHIISVG)is a specific antisense targeting peptide designed for the extracellular fragment EL2 of the LAPTM4B receptor.It was confirmed that this peptide could specifically recognize the LAPTM4B receptors on the membrane of HCC and then internalize into the cell.In this project,we will attempt to develop the novel fluorescent and PET tracers by labeling AP2H peptide with FAM and positron nuclide emitter of 18F or 68Ga.After that,we will investigate their receptor targeting and imaging potential in vivo using the fluorescent and micro PET/CT imaging.If our research can be successful,the new tracers will play an important role in the tumor diagnosis and help to guide the treatment.Objects:1.To synthesize AP2H peptide and then develop it to become a fluorescent probe(FAM-AP2H)by labeling it with 5-carboxyfluorescein(FAM).After that,to determine the uptake of FAM-AP2H in vitro and in vivo.2.To investigate how to radiolabel AP2H with 18F and 68Ga and obtain the corresponding PET probes.After that,to verify their capacity for invasively visualizing the receptor expression in vivo using micro PET/CT.3.Explore how to reduce liver and gallbladder excretion and improve the micro PET/CT imaging of mouse.Materials and Methods:All chemicals(reagent grade)were obtained from commercial suppliers and used without further purification.The AP2H peptide[IHGHHIISVG],FAM-AP2H peptide[FAM-IHGHHIISVG]and GGGRDN-AP2H peptide[GGGRDN-IHGHHIISVG]were purchased from ChinaPeptides Co.,Ltd.Shanghai,China).NODA-Mp-C6-AP2H was purchased from Chinesepeptide Co.,Ltd.(Hangzhou,China).1.Synthesis and Purification of AP2H Polypeptide(IHGHHIISVG)(1).Synthesis of AP2H Polypeptide(IHGHHIISVG)AP2H peptide was synthesized using standard solid phase peptide chemistry from Fmoc-protected amino acids.Symmetric anhydride method was used for by coupling of the first amino acid at the carboxyl terminus to 2-Chlorotrityl chloride resin.At the end of the reaction,sufficient pyridine and acetic anhydride were added to block remained active site on the resin.Amide bond coupling of next amino acid was achieved with HOBt/HBTU/DIEA as the peptide coupling reagent and repeated until the last amino acid was coupled to peptide resin.After the reaction was complete,using triflouoroacetic acid as main cleavage reagent to harvest the peptide from the resin.Removal of the solvent by rotary evaporation gave a crude oil that was triturated with cold ether.The crude mixture obtained was then centrifuged,the ether was removed by decantation,and the resulting white solid was purified by high performance liquid chromatogram(HPLC).The product was isolated by lyophilization and characterized by electrospray mass spectrometry.2.Labeling the AP2H with 5-carboxyfluorescein(FAM)5-FAM contains a carboxylic acid that can be used to react with primary amines via carbodiimide activation of the carboxylic acid.FAM was labeled with the amino group of first Isoleucine.After the completion of the labeling,the resulting solid was purified by HPLC.The product was isolated by lyophilization and characterized by electrospray mass spectrometry.3.In vitro binding test of FAM-AP2H(1)cell cultureHepG2 and BEL-7402 cell lines,reported to have over-expression of LAPTM4B receptor,was purchased from Institute of Biochemistry and Cell Biology,Shanghai or ATCC cell bank.L-02 cell lines,reported to have negative-expression of LAPTM4B receptor,was purchased from Institute of Biochemistry and Cell Biology,Shanghai cell bank.Cells were cultured in RPMI 1640 or DMEM containing 10%fetal bovine serum and 1%antibiotic at 37℃ in a humidified 5%carbon dioxide-containing atmosphere.(2)immunofluorescence to identify LAPTM4B positive and negative cells.HepG2、BEL-7402、L-02 cell lines,at a density of about 1 x 104cells/well seeded into 24-well plate.After overnight incubation,Washed with PBS twice,and cells were fixed with 4%of paraformaldehyde for 15min.Again washed with PBS twice.The cells were then incubated in 10%BSA for 1 hour to block nonspecific binding.discarded the liquide,followed by overnight incubation at 4 ℃ with dilution anti-LAPTM4B polyclonal antibody(Abcam,Shanghai,China)at a concentration of 5 μg/ml(diluted in Immnol Staining Primary Antibody Dilution Buffer,Beyotime,Shanghai,China).After washed with PBS three times,and incubated for 1 hour with a secondary goat anti-rabbit IgG fluorescent(CY3)conjugated antiboday(diluted 1:500 in Immnol Fluorence Staining Secondary Antibody Dilution Buffer,Beyotime,Shanghai,China).Follow by washed with PBS at least three times,cells were staining with DAPI solution for 5min,and then visualized by an Olympus Ix71 fluorescence microscope(Olympus,Japan).(3)In vitro imaging of combining capacity and competitive inhibition experiment of FAM-AP2H peptides with cells.Binding experiment:HepG2 or L-02 cell lines,in a density of about 1 × 104 cells/well seeded in confocal plate and incubator overnight.After washed twice with PBS and incubated with different concentration(0μM、2.5μM、5μM、10μM、20μM、40μM、80μM)of FAM-AP2H peptide at 4℃ for 1 hr.After rinse with PBS for three times,add 4%PFA 15min to fix cells.Follow by washing with PBS at least three times,cells were staining with DAPI solution for 5min,and then visualized by confocal microscopy(OLYMPUS,FLUOVIEW,FV1000)or Olympus Ix71 fluorescence microscope(Olympus,Japan).Quantitation:HepG2 cells with high expression of LAPTM4B receptor were used as receptor positive cells.HepG2 cells(1×106 cells)were placed in a flow cytometer for use.And incubated with different concentration of FAM-AP2H(OμM、2.5μM、5μM、10μM、20μM、40μM、80μM)at 4 ℃ for 1 hour.The unbound FAM-AP2H was washed by centrifugation at least 6 times.Using flow cytometer for detection(Beckman Coulter,MoFlo XDP).Competitive inhibition experiment:HepG2 cells were incubated with a mixed peptide solution at 4 ℃ for 1 hr.The concentration of peptide FAM-AP2H was maintained at 20μM,and AP2H peptide was gradually increased as OμM,200μM,400μM.(4)AP2H and FAM-AP2H cytotoxicity testHepG2 cells were cultured in log phase and plated on 96-well plates.The number of cells per well was about 2 × 103.After incubation overnight,100 μl AP2H(or FAM-AP2H)peptide(0 μM,20 μM,40 μM,80 μM)diluted with whole medium.After incubation for 24 h,lOul of cell counting kit-8(CCK-8,Dojindo Molecular Technologies,Inc.,Xiongben,Japan)solution was added each well for 1 hour,followed by light absorbance measurement at a wavelength of 450 nm.4.Tumor modal and verifity(1)To produce HepG2 and BEL-7402 tumors,a total of 1 × 106 cells were injected intramuscularly into the left flank of BALB/C athymic nude mice(purchased from the Laboratory Animal Center of the Southern Medical University).Tumor isografts were monitored until tumor size was≈1 cm in largest diameter after 4-6 weeks later.(2)To confirm the successful establishment of the tumor modal,the histopathologic examination was performed.HepG2 and BEL-7402 xenograft model was sacrified and the tumor was removed.Tumor tissue was collected and fixed with formalin.Tissue sections of 3μpm thinkness were prepared and placed on the clean glass slide.HE staining and immumohistochemical staining was performed routinely.5.Fluorescent Imaging of FAM-AP2H in the tumor modal(1)Fluorescent biodistribution in the HepG2/BEL-7402 xenograft modelHepG2/BEL-7402 tumor-bearing nude mice were intravenously injected with 150μl of ImM FAM-AP2H peptides.After 1hour or 5hour circulation,mice were sacrificed and organs were removed.After washing with the saline several times,the tumor and the normal organs were collected.The uptake of FAM-AP2H in the tumor and normal organs were observed under blue light with exposure times of 30 s,using the Kodak in-Vivo Imaging System F(Kodak,American)and processed for fluorescence intensity analysis.(2)Fluorescence images analysisUsing the Kodak MI analysis software,the regions of interest(ROI)were drawn around the border of the tumor or normal organs on the fluorescence images and fluorescence intensity was measured.The tumor/non-tumor ratios(T/NT ratios)were calculated by dividing the fluorescence intensity in the tumor to that of the normal organs.6.Synthesis of PET molecular probe of[18F]AlF-NODA-MP-C6-AP2H、[18F]-FP-AP2H、[68Ga]Ga-NODA-MP-C6-AP2H、[18F]-FP-GGGRDN-AP2H and in vivo PET/CT imaging(HepG2/BEL-7402)1.Synthesis of PET molecular probe of[18F]A1F-NODA-MP-C6-AP2H、[18F]-FP-AP2H、[68Ga]G-=NODA-MP-C6-AP2H、[18F]-FP-GGGRDN-AP2H1)Synthesis of PET molecular probe of[18F]AlF-NODA-MP-C6-AP2HUsing the method reported by laverman,P[32],NODA--MP-C6-AP2H was labeled with 18F.The peptide 0.25 mg was dissolved in 40 μl of pure wate,2ml of AlCl3(1.6ug),5ul of glacial acetic acid;324ul of acetonitrile,add 40ul 18F,100 degrees Celsius heating l0min.After cooling slightly,the column was washed with 15 ml of water,10 ml of 1X PBS,20 ml of water,400 ul of 10 mM hydrochloric acid in ethanol to give the product.2)Synthesis of PET molecular probe of[18F]-FP-AP2H and[18F]-FP-GGGRDN-AP2HSep-Pak QMA column adsorption accelerator bombardment out of the 18F,using the method reported by Hu,Kongzhen[34].3)Synthesis of PET molecular probe of[68Ga]Ga-NODA-MP-C6-AP2H68Ga was obtained by a 68 gallium/68 germanium germanium leaching.NODA-MP-C6-AP2H polypeptide 0.25 mg was diluted with 1 ml of HEPES liquid,1.25 M NaOAc,pH=4,and heated in a water bath,40℃ for 10 min.2.Quality control of[18F]AlF-NODA-MP-C6-AP2H,[18F]-FP-AP2H,[68Ga]Ga-NODA-MP-C6-AP2H、[18F]-FP-GGGRDN-AP2HThe chemical purity and radiochemical purity were determined by high performance liquid chromatography(HPLC).The radioactivity at different time points was determined by activity meter.The half-life and nuclear purity were determined by semi-logarithmic plot method.The radiochemical purity of the injection was measured at room temperature for 240 minutes.The stability of the marker in vitro was observed.3.micro PET/CT imaging(1).Micro-PET/CT scan was performed on a SIEMENS Inveon scanner(Siemens,Germany).Binding experiment:About 150μCi of of Radiolabeled peptide was intravenously injected into each mouse.Micro PET/CT images were acquired as 10-min static images at 60min after the injection with the mice under anesthesia.The images were reconstructed by a 3-dimensional ordered subsets expectation maximum(OSEM)algorithm and CT correction was necessary for attenuation correction.For the blocking experiment,mice bearing HepG2/BEL-7402 tumors were scanned(10 min static)at 1 hour after the coinjection of 150μCi of 18F-FP-GGGRDN-AP2H with 10 mg/kg AP2H peptide per mouse.(2).Biodistribution studiesUsing the Syngo analysis software,the regions of interest(ROI)were drawn around the border of the tumor or normal organs on the images of micro PET/CT and the radioactivity was measured.The results were presented as percentage injected dose per gram of tissue(%ID/g).Mean uptake(%ID/g)for a group of animals was calculated with standard deviations.The tumor/non-tumor ratios(T/NT ratios)were calculated by dividing the radioactivity in the tumor to that of the normal organs.7.The micro-PET/CT studies of U87MG xenograft modelCell culture,tumor model establishment,immunohistochemistry and micro PET/CT imaging are similar to the previous stepsStatistical AnalysisThe descriptive data were expressed as mean ±SD.Statistical Package for the Social Sciences,version 19.0(SPSS Inc.),was used for statistical analysis.Student’s t test or one-way anova was used for the statistical comparison between different groups.P values less than 0.05 were considered statistically significant.Results.1.Synthesis of the LAPTM4B-targeting peptide AP2H and FAM-AP2H,and identify its combining capacity with HepG2 cells1).Synthesis of the LAPTM4B-targeting peptide AP2HThe obtained AP2H peptide by solid phase synthesis showed as the white powder.The molecular mass(m/z:Da)determined by Mass chromatographic analysis(ESI)was 1069.7,which was in accordance with calculated 1069.24.It indicated LAPTM4B-targeting peptide of AP2H was correctly synthesized.The final product was purified by preparative HPLC.The purity of AP2H peptide was 98.89%%as determined by analytical HPLC.2).Synthesis of FAM-AP2HFAM was attached through its carboxylic acid which was reacted with the amino group of 1th Isoleucine of AP2H peptide and control peptide(sequence:IHGHHIISVG)via carbodiimide activation.The final products showed as pallide-flavens solid powder.The molecular mass(m/z:Da)determined by Mass chromatographic analysis(ESI)was 1427.9,which was in accordance with calculated1427.56.It indicated FAM-AP2H were correctly conjugated.The final products were purified by preparative HPLC.The purity of AP2H peptide were 98.23%as determined by analytical HPLC.3).Synthesis of NODA-MP-C6-AP2HThe obtained NODA-MP-C6-AP2H peptide showed as the white powder.The molecular mass(m/z:Da)determined by Mass chromatographic analysis(ESI)was 1574.87,which was in accordance with calculated.It indicated LAPTM4B-targeting peptide of AP2H was correctly synthesized.The final product was purified by preparative HPLC.The purity of NODA-MP-C6-AP2H peptide was 97.0%as determined by analytical HPLC.4).Expression of LAPTM4B receptor in different cellsConfocal imaging showed that LAPTM4B receptor was highly expressed in HepG2/BEL-7402 cells,but L-02 cells were negative.5).In vitro imaging of combining or saturation experiment of FAM-AP2H peptides with HepG2/L-O2 cells The results showed that HepG2 cells had high uptake of FAM-AP2H,with the increase of concentration,uptake of FAM-AP2H in HepG2 cells seemed to increase slightly.With a certain concentration as 80μM,the fluorescence intensity is saturated.The results are consistent with the saturation curve of receptor-ligand binding.L-02 cells did not show significant green fluorescence.The cells which treated with PBS as a control did not show any green fluorescence.Competitive inhibition experiment:The uptake of FAM-AP2H was significantly reduced when the concentration of the AP2H peptide was more than 10 times higher than FAM-AP2H.The results are consistent with the receptor-ligand binding characteristics.6).Both AP2H and FAM-AP2H showed no cytotoxicity and were suitable for fluorescence imaging.2.Fluorescent imaging of FAM-AP2H in tumor modal(1).Establishment of tumor model in nude mice(HepG2/BEL7402)HepG2 cells were inoculated subcutaneously in 4-week-old athymic nude mice,and the tumor nodules were seen after 14 days.After 6 weeks,the tumors were increased to 1.0 cm for the test.A total of 30 mouse were inoculated subcutaneously.Except 1 had no tumor growth,others had succeeded.The rate of tumor formation was 96.7%.In order to verify whether the tumor model meet the requirements or not,one of the tumors was resect,and finished HE staining and immunohistochemical staining,pathological findings showed LAPTM4B receptor was positive.BEL-7402 cells were inoculated subcutaneously in 4-week-old athymic nude mice,and the tumor nodules were seen after 14 days.After 6 weeks,the tumors were increased to 1.0 cm for the test.A total of 30 mouse were inoculated subcutaneously.The rate of tumor formation was 100.0%.In order to verify whether the tumor model meet the requirements or not,one of the tumors was resect,and finished HE staining and immunohistochemical staining,pathological findings showed LAPTM4B receptor was positive.(2).The fluorescent biodistribution in the HepG2/BEL7402 xenograft modelThe study demonstrated that FAM-AP2H was intensely uptaked by the tumor,where as the uptake of FAM-AP2H in the brain was minimal.And the fluorescence intensity of tumor in 5h was significantly higher than 1h.T/NT ratios were 1.68 ±0.48(tumor/liver),1.36 ± 0.35(tumor/brain),2.07 ± 0.67(tumor/heart),1.75 ±0.40(tumor/lung),2.18 ± 0.64(tumor/speen),0.93 ± 0.08(tumor/kidney),0.51 ±0.28(tumor/intestine),1.63 ± 0.42(tumor/muscle),respectively.For BEL-7402 models,T/NT ratios were 1.36±0.21(tumor/liver),1.28±0.15(tumor/brain),1.75± 0.54(tumor/heart),1.61 ± 0.18(tumor/lung),1.96 ± 0.30(tumor/speen),0.96 ±0.17(tumor/kidney),0.39 ± 0.20(tumor/intestine),1.91 ± 0.73(tumor/muscle),respectively.The present study showed that the fluorescent distributions in the gallbladder,intestine and kidneys were very high which indicated that FAM-AP2H excreted from the body through the hepato-biliary tract and urinary system.The fluorescent intensity in the liver after 5 h circulation was high,which suggested that washout of FAM-AP2H in the liver was slow and FAM-AP2H was uptaked by the hepatocellular cells.The uptake of FAM-AP2H in other organs,such as heart,lungs,spleen,muscle was very low.3.Development of PET molecular probes[18F]AlF-NODA-MP-C6-AP2H,[18F]-FP-AP2H,[18F]-FP-GGGRDN-AP2H,[68Ga]Ga-NODA-MP-C6-AP2H and in vivo micro PET/CT imaging(1)[18F]AlF-NODA-MP-C6-AP2HThe synthesis of[18F]AlF-NODA-MP-C6-AP2H was successfully carried out with reference to the chelating method of[18F]AlF.The total reaction time was 15 min.AP2H polypeptide 0.25mg with 40ul pure water dissolved in 2mlAxygen centrifuge tube,add 6ul,2mM Alcl3(1.6ug),5ul glacial acetic acid;324ul acetonitrile,;add 40ul 18F water(39.lmci),100 degrees Celsius heating 10min.After cool off,transfer to C18 column with 15ml water,rinse with 10ml 1X PBS,and then 20ml water washing,400 ul,10 mM hydrochloric acid in ethanol to give a 12.9 mci product.Yield 33.0%Micro PET/CT imaging studies of HepG2/BEL-7402 tumor showed that a large amount of radioactivity accumulated in the tumor site at 60 minutes after injection.The radioactivity uptake in the tumor was high.Intense radioactivity was found in the liver,gallbladder.In HepG2 tumor model,the radioactivity is 2.20±0.55%ID/g,tumor/liver=2.00 ± 0.52.In BEL-7402 tumor model,the radioactivity is 1.00±0.26%ID/g,tumor/liver=2.33 ± 1.15.(2)[18F]-FP-AP2H,The synthesis of[18F]-FP-AP2H was successfully carried out with reference to the literature.The total reaction time is 3-4 hour.The Yield with out Attenuation correction is 15%Micro-PET/CT imaging of HepG2/BEL-7402 tumor showed that at 60 minutes after injection,the radioactivity uptake in the tumor was high.Intense radioactivity was found in the liver,gallbladder.In HepG2 tumor model,the radioactivity is 3.70±0.56%ID/g,tumor/liver=0.81 ±0.06.(3)[68Ga]Ga-NODA-MP-C6-AP2H68Ga(6.24mCi)was added,and a total of[68Ga]Ga-NODA-MP-C6-AP2H 3.0mCi was synthesized by the chemical synthesis method previously described.The Yield with out Attenuation correction is 90%.Image analysis found that the tumor has high radioactive uptake,while the radiolabeled drug mainly metabolic through the liver-and kidney.Bladder and Kidney visible radioactive accumulation.In HepG2 tumor model,the radioactivity is 2.20 ±0.55%ID/g,tumor/liver=1.10±0.07.(4)[18F]-FP-GGGRDN-AP2HThe synthesis of[18F]-FP-GGGRDN-AP2H was successfully carried out with reference to the literature.The total reaction time is 3-4 hour.The Yield with out Attenuation correction is 15%Micro-PET/CT imaging of HepG2/BEL-7402 tumor showed that at 60 minutes after injection,the radioactivity uptake in the tumor was high.Intense radioactivity was found in the kidney.In HepG2 tumor model,the radioactivity is 1.70±0.27%ID/g,tumor/liver=1.71 ±0.33.In BEL-7402 tumor model,the radioactivity is 2.20 ±0.61%ID/g,tumor/liver=1.60 ± 0.13.4.PET/CT imaging of[18F]AlF-NODA-MP-C6-AP2H in U87MG glioma xenografts tumor modelThe expression of LAPTM4B receptor of U87MG cells was confirmed by immunohistochemistry.The U87MG tumor could uptake[18F]AlF-NODA-MP-C6-AP2H,which showed radioactivity.Central radiative defect was considered to be liquefaction necrosis.A large amount of radioactivity was found in the intraperitoneal cavity of the tumor-bearing mice,and the radioactivity of the lungs and cranial nerves was sparse.Tumor/brain ratio was20.74± 5.94.Conclusions:1.In the present study,we successfully synthesized a LAPTM4B targeting peptide of AP2H and have developed it to become the novel fluorescent and PET tracers by labeling chemistry.2.FAM-AP2H was confirmed to possess the capacity of specific binding with HCC cells and targeting to the tumors which highly expressed the LAPTM4B receptors by fluorescent test and imaging.3.Radiolabeled AP2H as three new PET tracers.In the[18F]-FP-AP2H,[18F]AlF-NODA-MP-C6-AP2H and[68Ga]Ga-NODA-MP-C6-AP2H probes,[18F]AlF-NODA-MP-C6-AP2H have best imaging performance.but the uptake of gallbladder and intestine were too high.4.Modified peptide GGGRDN-AP2H,and radiolabeled as[18F]-FP-GGGRDN-AP2H,The results showed that the hepatobiliary excretion of the modified probe was significantly decreased than[18F]-FP-AP2H,which could significantly increase the ratio of tumor/liver,tumor/gallbladder and tumor/intestine,and improved the imaging effect on the AP2H polypeptide.