| BackgroundRemoval of the glioma tumor completely and protection of the brain function as much as possible is cruicial for the goal of improving the patients’ survival and postoperative quality of life. However, the completeness of tumor removal is difficult because it is not easy to differentiate the tumor from normal tissue before and during the surgery. Developing the fluorescent and PET molecular probes would be of great value. Utilizing the high sensitivity of PET detective capacity and high specificity of probe tareting, it is suggest that we can delineate the infiltration of glioma more celearly before the operation by PET/CT imaging. Meanwhile, it is also suggest that we can "paint" the tumor with the fluorescent probe and guide the surgeon to removal the tumor completely as much as possible during the surgury by utilizing the high sensitivity of fluorescent probe for the differentiate the tumor from normal tissue.Neuropilin receptor (NRP) is a co-receptor for vascular endothelial growth factor (VEGF). It is over-expressed in the endothelial cell membrane of angiogenic vessels and the membrane of glioma cells. tLyP-1(sequence:CGNKRTR) is a novel NRP targeting peptide. It was reported that the tLyP-1phage bound to immobilized NRP1about120times more than insertless control phage. Development of the fluoresceit and18F Labeled tLyP-1peptide might have a potential to be the good tracers for imaging of glioma.Objective:1. To synthesis the NRP-targeting peptide of tLyP-1and identify its combining capacity with glioma cells, making preparation for the further targeting probe synthesis and tumor targeting imaging.2. To develop a novel fluorescent probe by labeling the tLyP-1with5-carboxyfluorescein (FAM), then verify its ability to "paint" the glioma tumor and assess its potential as a fluorescent molecular probe for glioma.3. To develop a novel PET molecular probe using18F-labeled tLyP-1peptide and to investigate its potential for the diagnosis and delineation of the glioma by PET/CT.Methods1. Synthesis the NRP-targeting peptide of tLyP-1and identify its combining capacity with glioma cells1).Synthesis of the NRP-targeting peptide of tLyP-1tLyP-1peptide 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 to2-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, triflouoroacetic acid as main cleavage reagent was used 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 tLyP-1and the control peptides with5-carboxyfluorescein (FAM)5-FAM contains a carboxylic acid that can be used to react with primary amines via carbodiimide activation of the carboxylic acid. Duing the synthesis of tLyP-1and control peptide (sequence:MAQKTSH), Fmoc-Lys (Dde)-OH was coupled to the peptide to form the4th Lys. After the completion of the synthesis of tLyP-1and control peptide. Dde in the4th Fmoc-Lys (Dde)-OH was removed and FAM was labeled with the amino group of lysine with HOBt/HBTU/DIEA as the coupling reagent. 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 and block test of FAM-tLyP-1with U87MG cellsU87MG cell lines, reported to have over-expression of NRP, was purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM containing10%fetal bovine serum at4℃in a humidified5%carbon dioxide-containing atmosphere.For fluorescence microscopy, U87MG cells were seeded on cover slips in16-well plates and incubated in DMEM/F-12(0.5ml/well) overnight. U87MG cells were incubated at4℃for1h with different concentration of0μM,1μM,4μM,10μM,20μM and40μM solution of a FAM-tLyP-1in PBS/1%BSA. For the blocking experiment, the U87MG cells cells of two wells were first treated with20μM non-conjugated tLyP-1peptide either and then incubated with1μM and4μM FAM-tLyP-1at4℃for1h. After that, the cells were washed three times with phosphate-buffered saline (PBS). Slides were visualized under a blue light using a fluorescent inversion microscope (Olympus IX71).4).Tumor modalTo produce U87MG tumors, a total of1×106cells (U87MG) 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≈1cm in largest diameter after4-6weeks later. To confirm the successful establishment of the tumor modal, the histopathologic examination was performed. U87MG glioblastoma xenograft model was sacrified and the tumor was removed. Tumor tissue was collected and fixed with formalin. Tissue sections of3μm thinkness were prepared and placed on the clean glass slide. HE staining was performed routinely.5).The tumor tissue fluorescent imagingU87MG tumor-bearing nude mice were intravenously injected with150μl of1mM FAM-tLyP-1peptides. After1h circulation, mice were killed and tumor and brain were removed. The tumor tissue sections were prepared and mounted in DAPI (4’,6-diamidino-2-phenylindole)-containing mounting media. After that, they were collected and viewed under blue light using an Olympus DP71fluorescent microscope (Olympus America, Center Valley, PA, USA).2. Fluorescent Imaging of tLyP-1in the tumor modal1). Fluorescent biodistribution in the U87MG glioblastoma xenograft modelU87MG tumor-bearing nude mice were intravenously injected with150μl of1mM FAM-tLyP-1peptides or150μl of ImM FAM-labeled control peptide. After1h circulation, mice were sacrificed and tumor and the normal organs were removed. After washing with the saline several times, the tumor and the normal organs were collected. The uptake of FAM-tLyP-1in the tumor and normal organs were observed under blue light with exposure times of60s, using the Kodak in-Vivo Imaging System F (Kodak, American) and processed for fluorescence intensity analysis.2). In-vivo fluorescent sectioned imaging of tumor87MG tumor-bearing nude mice were intravenously injected with150μl of1mM FAM-tLyP-1peptides. After1h circulation, mice were sacrificed and freezed under-80℃. The mice were cutted into the coronal sections with the cutting-off machine and was placed on the clean grass container. The uptake of FAM-tLyP-1in the tumor and normal organs were observed under blue light using the Kodak in-Vivo Imaging System F.3). 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.3. Synthesis of PET molecular probe of18F-tLyP-1and in vivo PET/CT imaging1) Preparation of N-succinimidyl-4-[18F]fluorobenzoate (18F-SFB).Ethyl4-(trimethylammonium triflate) benzoate (1)(5.0mg,20mmol) in anhydrous MeCN (1mL) was added to the dried K222/K [18F]F and the mixture heated at90℃for10min to produce ethyl4-[18F]fluorobenzoate (2). The ethyl ester was subsequently hydrolyzed to form (3) using20mL of tetrapropylammonium hydroxide (1.0M in water) at120℃for3min, and then the mixture azeotropically dried using MeCN (1mL). Subsequently, a solution of N,N,N0,N0-tetramethyl-O-(N-succinimidyl) uronium hexafluorophosphate (HSTU)(12mg,33mmol) in MeCN (1mL) was added and the solution heated at90℃for5min. After cooling,5%aqueous acetic acid (9mL) and water (15mL) were added. The reaction mixture was passed through a C18Sep-Pak cartridge, a Sep-Pak alumina cartridge and a Lichrolut SCX cartridge (200mg, Merck) in series. The Sep-Pak C18cartridge trapped the18F-SFB, the Sep-Pak alumina cartridge removed the free18F, and the SCX cartridge removed the impurities. The cartridges were washed with10%aqueous MeCN (15mL) and then the product18F-SFB eluted with MeCN (2mL).2). Synthesis of18F-tLyP-1as a PET/CT probe.18F-SFB was added to tLyP-1peptide (250μg) in800μL of sodium borate buffer (50mmol/L, pH8.5). The reaction mixture was gently mixed at40℃for30min. Final purification was accomplished by semi-preparative HPLC and the tracers were reconstituted in phosphate-buffered saline (PBS, pH7.4) and passed through a0.22μm Millipore filter (Millipore Corp.) for in vivo applications.3). PET/CT and microPET/CT imagingPET/CT scan was performed on a SIEMENS Biograph mCTx scanner (Siemens, Germany). Micro-PET/CT scan was performed on a SIEMENS Inveon scanner (Siemens, Germany). The PET/CT or micro-PET/CT studies of U87MG glioblastoma xenograft model were performed by tail-vein injection of3.7MBq (100μCi) of18F-tLyP-1. MicroPET/CT images were acquired as10-min static images at30min,60min or120min after the injection with the mice under isoflurane anesthesia. PET/CT images were acquired as10-min static images only at60min.The images were reconstructed by a3-dimensional ordered subsets expectation maximum (OSEM) algorithm and CT correction was necessary for attenuation correction.4). PET/CT and microPET/CT image analysisUsing the Syngo analysis software, the regions of interest (ROI) were drawn around the border of the tumor or normal organs on the images of PET/CT or microPET/CT and the radioactivity was measured. The tumor/non-tumor ratios (T/NT ratios) were calculated by dividing the radioactivity in the tumor to that of the normal organs.4. Statistical AnalysisThe descriptive data were expressed as mean±standard deviation. Statistical Package for the Social Sciences, version13.0(SPSS Inc.), was used for statistical analysis. The independent samples t test was used for the statistical comparison of T/non-NT ratios of FAM-tLyP-1and FAM-control peptide. The paired sample t test was used for the statistical comparison of T/non-NT ratios of F-tLyP-1microPET/CT between60min and120min after introvenuous injection. A P value of less than0.05was considered statistically significant.Results1. Synthesis of the NRP-targeting peptide of tLyP-1and identify its combining capacity with glioma cells1).Synthesis of the NRP-targeting peptide of tLyP-1The obtained tLyP-1peptide by solid phase synthesis showed as the white powder. The molecular mass (m/z:Da) determined by Mass chromatographic analysis (ESI) was834.9, which was in accordance with calculated833.97. It indicated NRP1-targeting peptide of tLyP-1was correctly synthesized. The final product was purified by preparative HPLC. The purity of tLyP-1peptide was99.65%as determined by analytical HPLC.2). Synthesis of FAM-tLyP-1and FAM labeled control peptideFAM was attached through its carboxylic acid which was reacted with the amino group of4th lysine of tLyP-1peptide and control peptide (sequence:MAQKRSH) via carbodiimide activation. The final products showed as pallide-flavens solid powder. The molecular mass (m/z:Da) determined by Mass chromatographic analysis (ESI) was1192.8and1213.8, respectively, which was in accordance with calculated1192.29and1215.33respectively. It indicated FAM-tLyP-1and FAM labeled control peptide were correctly conjugated. The final products were purified by preparative HPLC. The purity of tLyP-1peptide and the FAM labeled control peptide were98.01%and98.4%respectively as determined by analytical HPLC.3).In vitro imaging of combining capacity of FAM-tLyP-1peptides with U87MG cellsU87MG cells were incubated with different concentration of FAM-tLyP-1peptide at4s℃for1hr. The present study showed that FAM-tLyP-1can be strongly uptaked by the U87MG cells, even at a very low concentration of1μM. The uptake of FAM-tLyP-1in U87MG cells seemed to increase slightly with the increase of concentrations. The cells which treated with PBS as a control did not show any green fluorescence. This result suggested that FAM-tLyP-1can be intensely uptaked by the U87MG cells.For the blocking experiment, the cells of two wells were first treated with20μM non-conjugated tLyP-1peptide either and then with1μM and4μM FAM-tLyP-1. The study showed that the uptake of FAM-tLyP-1in U87MG cells was dramatically inhibited by incubation with excessive quantity of non-conjugated tLyP-1peptide. The FAM-tLyP-1uptake in U87MG cells blocked by20times excessive quantity of non-conjugated tLyP-1peptide was lower than that of5times block. The result suggested that FAM-tLyP-1and non-conjugated tLyP-1peptide have the relationship of competitive inhibition, which is on the accordance with the principle of the receptor-ligand competitive binding. 4).Tumor modalThe tumor models were generated by intramuscularly injection of U87MG cells into the left flank of BALB/C athymic nude mice. Fourteen days after inoculation, the tumors were visualized. At4-6weeks after inoculation, the tumors were found to grow up to about1.0cm in diameter. There were6groups of tumor modals generated in different time with5mice in each group. The achievement ratio of tumor modal generation was80%(24/30).The tumor of the above animal modal was confirmed to be the glioma by the histopathologic examination.5).The tumor tissue fluorescent imagingAfter1hr circulation of FAM-tLyP-1peptides in the U87MG tumor-bearing nude mice, the mice were sacrificed and the tumor tissue fluorescent imaging was performed. The confocal microscope images showed FAM-tLyP-1was mostly accumulated in the angiogenic blood vessels wall at1hr after intravenous injection of FAM-tLyP-1. No FAM-tLyP-1was seen to be uptaked by the tumor cells in this time period.At4h after intravenous injection, FAM-tLyP-1was still mostly accumulated in the angiogenic blood vessels wall. However, in this time period, slightly uptake of FAM-tLyP-1was found in the tumor cells close to the angiogenic blood vessels.The present study indicated that the main coaggregation sites of FAM-tLyP-1in the tumor is the angiogenic blood vessels, but not the tumor cells. The high intensity of FAM-tLyP-1binding with the endothelial cell of the angiogenic blood vessels may influence the permeation of FAM-tLyP-1into tumor parenchyma and binding with the tumor cells.FAM-tLyP-1uptake in the normal brain tissue was found to be very low and the distribution of fluorescence was not in the blood vessels. The low uptake of FAM-tLyP-1in the brain was suggested to be benefit for the detection of gliomas.2. Fluorescent imaging of FAM-tLyP-1in the tumor modal1). The fluorescent biodistribution in the U87MG xenograft modelAfter1h circulation of FAM-tLyP-1in the U87MG tumor-bearing nude mice. The mice were sacrified and the fluorescent biodistribution of FAM-tLyP-1was studied. The study demonstrated that FAM-tLyP-1was intensely uptaked by the tumor, whears the uptake of FAM-tLyP-1in the brain was minimal. The uptake in the tumor was significantly higher than that in the normal brain with Tumor/brain ratios was3.44±0.83. Meanwhile, except those of kidney and intestine, T/NT ratios of other organs were all larger than2.0. The present study showed that the fluorescent distributions in the gallbladder, intestine and kidneys were very high which indicated that FAM-tLyP-1excreted from the body through the urinary system and hepato-biliary tract. The fluorescent intensity in the liver after1hr circulation was low, which suggested that washout of FAM-tLyP-1in the liver was fast and FAM-tLyP-1was not uptaked by the hepatocellular cells. The uptake of FAM-tLyP-1in other organs, such as heart, lungs, spleen, muscle was also very low.On the contrary to FAM-tLyP-1, the present study showed only slightly uptake of control peptide was found in the tumor after lhr circulation. The fluorescent distribution of control peptide in the tumor was slightly higher than that of normal brain with the tumor/brain ratios of1.32±0.15, which was significantly lower than that of FAM-tLyP-1(t=-5.547,P=0.001)。2). In-vivo fluorescent sectioned imaging of tumorIn order to confirm the findings in the fluorescent biodistribution study, in-vivo fluorescent sectioned imaging of tumor was performed after1h circulation of FAM-tLyP-1in the U87MG tumor-bearing nude mice. In-vivo fluorescent sectioned imaging showed FAM-tLyP-1accumulated intensely in the tumor. However, no fluorescence was found in the brain, heart, lungs, liver, muscle and bone. The fluorescence in the kidneys and intestine were very intense. The result of the in-vivo fluorescent sectioned imaging of the U87MG tumor modal was coincident with that of the fluorescent biodistribution study.These results demonstrated FAM-tLyP-1can selectively target to and "paint" the glioma tumor. Thus, FAM-tLyP-1has a potential to become a good fluorescent tracer for imaging of glioma. 3. Synthesis of PET molecular probe of18F-tLyP-1and in vivo PET/CT imaging1) Synthesis of PET molecular probe of18F-tLyP-1Using the method reported by Chen xiaoyuan,[Lys4] tLyP-1was labeled with18F by coupling the Lys4amino group with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) under slightly basic condition (pH8.5).The total reaction time, including final HPLC purification was about250min. The overall radiochemical yield without decay correction was8-12%. The radiochemical purity of the labeled peptides was larger than98%according to analytic HPLC.2) The in-vivo PET/CT and microPET/CT scanThe in-vivo microPET/CT imaging of subcutaneous U87MG xenograft model was performed at30min,60min and120min after injection of18F-tLyP-1.The study showed18F-tLyP-1accumulated a lot in the tumor at30min after injection. The radioactivity distribution in the heart, liver, intestine, kidney and the bladder were very high. The radioactivity in the blood pool was also high. No obviously radioactivity was found in the brain, lung and muscle. At60min and120min after injection, the radioactivity uptake in the tumor was also very high. The radioactivity in the heart and liver decreased to be the low background level. Intense radioactivity was found in the gallbladder at60min and120min after the injection. Intense radioactivity was also seen in the intestine, kidney and the bladder. At1h after injection, the radioactivity in the the blood pool decreased quickly and the tumor was visualized clearly. The Tumor/brain ratios at the time point of60min and120min were2.98±0.52vs.3.25±0.69(t=-0.956, P=0.393)The in-vivo PET/CT imaging of subcutaneous U87MG xenograft model was performed at60min after injection of18F-tLyP-1.The radioactivity biodistribution of18F-tLyP-1seen on the images of PET/CT was similar to that on the images of microPET/CT and the tumor could also be visualized clearly. However, the quality of microPET/CT image was much better than that of PET/CT.The microPET/CT and PET/CT imaging indicated that18F-tLyP-1can target to the tumor. Uptake of18F-tLyP-1in the tumor was very intense with the high tumor/brain ratios. Thus,18F-tLyP-1showed a potential to become a good PET tracer for imaging of glioma.The radioactivity distribution in the gallbladder, intestine, kidneys and urinary bladder were very high, which indicated that18F-tLyP-1was also excreted through the urinary system and hepato-biliary tract. High radioactivity distribution in the abdomen will hinder the ability of this tracer to detect the tumor in this region.The biodistribution of18F-tLyP-1was similar to that of FAM-tLyP-1.Conclusions:1. In the present study, we successfully synthesized a NRP targeting peptide of tLyP-1with a high purity of99.03%.2. We successfully synthesized a fluorescent molecular probe by labeling the tLyP-1with5-carboxyfluorescein (FAM) with a high purity of98.01%.3. In vitro imaging study confirmed that FAM-tLyP-1can be intensely uptaked by the U87MG glioma cells. The uptake of FAM-tLyP-1in the U87MG cells was in accordance with the principle of the receptor-ligand competitive binding.4. The tumor tissue fluorescent imaging study indicated the main coaggregation sites of FAM-tLyP-1in the tumor is the angiogenic blood vessels, although uptake of FAM-tLyP-1was also found in some tumor cells.5. Fluorescent imaging of tLyP-1in the tumor modal demonstrated FAM-tLyP-1can selectively target to and "paint" the glioma tumor with the high Tumor/brain ratios. FAM-tLyP-1showed a potential to become a good fluorescent tracer for imaging of glioma.6. The fluorescent biodistribution study indicated that FAM-tLyP-1was excreted through the urinary system and hepato-biliary tract. High fluorescent distribution in the abdomen will hinder the ability of this tracer to detect the tumor in this region.7. We successfully synthesized a PET molecular probe of18F-tLyP-1by labeling the tLyP-1with positron emitter of18F with a high purity of98.0%.8. The in-vivo PET/CT and microPET/CT scans indicated that18F-tLyP-1can target to the tumor. Uptake of18F-tLyP-1in the tumor was very intense with the high T/B ratios. The biodistribution of18F-tLyP-1was similar to that of FAM-tLyP-1.18F-tLyP-1showed a potential to become a good PET tracer for imaging of glioma. |
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