Systhesis And Evaluation Of Novel D Type Peptide Probes Targeting To Neuropilin-1 Receptor For PET Imaging

Neuropilin-1（NRP-1）is a type I transmembrane glycoprotein and a co-receptor of vascular endothelial growth factor,which can be found in a variety of endothelial cells of the neovascularization.It can promote VEGF functions mediated by VEGFR-2,including angiogenesis,activation,proliferation,and migration of endothelial cells.Bergé and other scholars have also proved that NRP-1 widely over-expressed in various human neoplasms,such as glioma,prostate cancer,pancreatic cancer,gastric cancer,colorectal cancer,breast cancer,non-small cell lung cancer,oral squamous cell carcinoma,etc.On the contrary,NRP-1 exists in many normal tissues with low levels.These suggest that NRP-1 may be potentially a potential target for tumor imaging.Previously,we had developed a NRP-1 targeting peptide,named tLyP-1,to be NRP-1 PET and fluorescent molecular probes by labeling it with positron emitter of 18F and FAM successfully,which could visualize NRP-1 positive glioma lesions in vivo.However,the probes have obvious hepatobiliary excretion that hamper the detection of liver lesions.Meanwhile,the uptake of probes in the tumors was not high enough.With the development of targeting peptide of NRP-1,our team attempt to design and explore novel probe to make up the shortcomings of tLy-1 probes and impove the imaging quality for tumor.A new NRP-1 targeting peptide,named LA7R,was screened by Binetury-Tournaire through phage display technology in 2000,which was reported to have a high affinity for NRP-1 and VEGFR-2 but showed instability in vivo.A improved peptide（Sequence:rpplwta）,which made up of D-amino acids with a reversed sequence of LA7R,was introduced by Man Ying in 2018 and was suggested to be able to resistant to the lysis of peptidase in the blood circulation.Thus,it can keep stability and target ability in vivo.However,there is no literature about converting it into PET molecular probe.In the present study,we modified this peptide with NOTA and attempt to develop it to be a novel targeting PET probe ¹⁸F-AlF-NOTA-rpplwta by using ¹⁸F-AlF chelation method.In addition,we also designed a novel targeting probe ¹⁸F-AlF-NOTA-atwlppr,which is D type peptide with a same sequence of LA7R（Sequence:atwlppr）.We then explored the imaging capabilities of both novel PET probes for NRP-1 positive tumor imaging in vivo and hope to overcome the shortcomings of tLy-1 probes.ObjectivesTo develop two novel PET probes targeting to NRP-1 using two D type peptides of LA7R and evaluate whether they can improve the imaging efficiency of the tumor and reduce the normal background radioactivity in the liver.Methods1.D-amino acid of LA7R（atwlppr and rpplwta）,and their NOTA-modified samples（NOTA-atwlppr and NOTA-rpplwta）were synthesized,isolated,purified and identified.2.Determining the affinities of NOTA-atwlppr and NOTA-rpplwta to NRP-1 protein using surface plasmon resonance（SPR）measurements,and compare their affinities to the original ones（atwlppr and rpplwta）.3.Radiolabeling the probes of ¹⁸F-AlF-NOTA-atwlppr and ¹⁸F-AlF-NOTA-rpplwta and evaluate their labeling yields and radiochemical purities.4.Measuring the Octanol/Water partition coefficients,in vivo stability and in vitro stability of two PET probes.5.Measuring the cell uptake and efflux to assess the binding capability of two PET probes to NRP-1 positive U-87MG cells.6.The NRP-1 positive U-87MG tumor models were established.Micro PET/CT was performed to evaluate the tumor detective capability,the bio-radioactivity distribution of ¹⁸F-AlF-NOTA-atwlppr and ¹⁸F-AlF-NOTA-rpplwta,and the effect of saturation suppression on uptake of two PET probes in the tumors using excessive amount of cold atwlppr and rpplwta as inhibitors.7.Immunohistochemical staining was used to determine the NRP-1 expression in the U-87MG tumor tissues.Results1.The peptides of atwlppr,rpplwta and their NOTA-modified ones（NOTA-atwlppr and NOTA-rpplwta）were synthesized using the method of solid-phase chemical synthesis.Mass chromatographic analysis showed that the molecular mass（MS）（m/z:Da）of produced atwlppr,rpplwta,NOTA-atwlppr and NOTA-rpplwta were in accordance with their calculated MS,indicating the synthesis were successful.All the samples were proved to have high purity（purity>95.0%）after purified by preparative HPLC.2.SPR assay revealed that the dissociation constants（KD）of atwlppr,rpplwta,NOTA-atwlppr and NOTA-rpplwta to NRP-1 were 1.08×10-8mol,2.67×10-8mol,1.92×10-8mol and 3.22×10-8mol respectively.Both D type peptides and their NOTA modified ones were similar in affinity,suggesting that NOTA modification produce had minimal damage to the binding affinity.3.¹⁸F-AlF-NOTA-atwlppr and ¹⁸F-AlF-NOTA-rpplwta were successfully radiolabeled by using the ¹⁸F-AlF chelate chemistry with the yields of 78.2313.50%and 77.13±3.65%respectively.The radiochemical purifies reached 98.70±1.23%and 98.38±1.07%respectively after purified by HPLC.Each product had a single peak,which was at the nearly same peak position of the intact peptide.4.Octanol/Water partition coefficient（Logp）of ¹⁸F-AlF-NOTA-atwlppr and ¹⁸F-AlF-NOTA-rpplwta were-2.90±0.03 and-2.80±0.04 respectively,which indicated both probes were hydrophilic and there was no significant difference between them（P>0.05）.5.¹⁸F-AlF-NOTA-atwlppr and ¹⁸F-AlF-NOTA-rpplwta showed stable in vitro and in vivo.In vitro,after incubating with PBS and serum after incubation at 37℃for 2 hours,98.69±1.01%and 98.63±1.36%of intact ¹⁸F-AlF-NOTA-atwlppr and 99.26±1.18%and 99.13±1.51%of intact ¹⁸F-AlF-NOTA-rpplwta were detected.In vivo,intact radioactivity of ¹⁸F-AlF-NOTA-atwlppr was found 97.31±1.58%in the U-87MG tumor,96.82±1.73%in the blood,86.19±2.42%in the liver and 53.25±2.94%in the kidneys respectively at 1h after intravenously administration.Similarly,intact radioactivity of ¹⁸F-AlF-NOTA-rpplwta was found 93.70±1.74%in the U-87MG tumor,91.54±1.03%in the blood,82.43±2.61%in the liver and 51.05±2.03%in the kidneys.There was no significant difference between them（P>0.05）.6.Cellular uptake assays revealed that the uptake of ¹⁸F-AlF-NOTA-atwlppr was similar to that of ¹⁸F-AlF-NOTA-rpplwta in U-87MG cells.Two PET tracers all reached the maximum uptake value at 120min.7.Micro PET/CT showed the U-87MG tumors could be clearly visualized by ¹⁸F-AlF-NOTA-atwlppr and ¹⁸F-AlF-NOTA-rpplwta at 30min after intravenously injection although the background radioactivity in the blood and heart were high.Tumors could still be visualized clearly with high uptake of probes at 60 min and 120 min.The radioactivity uptake in U-87MG tumors were 4.83±1.17%ID/g,3.60±1.15%ID/g and 1.73±0.11%ID/g for ¹⁸F-AlF-NOTA-atwlppr and 5.20±0.26%ID/g,4.10±0.69%ID/g and 2.20±0.56%ID/g for ¹⁸F-AlF-NOTA-rpplwta at 30min,60min and 120min after injection.After inhibited with excessive amount of atwlppr and rpplwta,the uptake of radioactivity in the tumors decreased significantly with 1.30±0.44%ID/g,1.04±0.58%ID/g and 0.79±0.39%ID/g for ¹⁸F-AlF-NOTA-atwlppr and 1.59±0.18%ID/g,1.18±0.16%ID/g,0.90±0.03%ID/g for ¹⁸F-AlF-NOTA-rpplwta,respectively at 30min,60min and 120min after injection,which were all significantly lower than those without inhibition（P<0.05）.The uptake of probes in the brain was low,especially ¹⁸F-AlF-NOTA-atwlppr,with 0.27±0.19%ID/g for ¹⁸F-AlF-NOTA-atwlppr and 0.90±0.35%ID/g for ¹⁸F-AlF-NOTA-rpplwta at 60min.As a result,high tumor/brain ratios were achieved with 15.91±6.74 for ¹⁸F-AlF-NOTA-atwlppr and 4.83±1.36 for ¹⁸F-AlF-NOTA-rpplwta（P<0.05）.The radioactivity uptake in the liver of two probes were medium with 1.29±0.44%ID/g for ¹⁸F-AlF-NOTA-atwlppr and 2.10±0.20%ID/g for ¹⁸F-AlF-NOTA-rpplwta.Higher tumor/liver ratio of ¹⁸F-AlF-NOTA-atwlppr（2.80±0.10）was observed comparing to that of ¹⁸F-AlF-NOTA-rpplwta（1.95±0.20）（P<0.05）.Both probes showed much high radioactivity in the kidneys and persisted high radioactivity accumulation in gallbladder,suggesting that the urinary system and the hepatobiliary system were the excretory pathways,especially the former one.Minimal uptake of probes in the bone implied that no defluorination occurred in the body.8.Immunohistochemical staining demonstated that NRP-1 was highly expressed in U-87MG tumor.Conclusion1.The ¹⁸F-AlF-NOTA-atwlppr and ¹⁸F-AlF-NOTA-rpplwta were successfully prepared by A118F chelation with RCP up to 97%.The radiolabeling is simple,time-saving,and can be completed by the "one-pot method".It can be carried out by modular automated production,which will be easily transferred to clinic.2.¹⁸F-AlF-NOTA-atwlppr and ¹⁸F-AlF-NOTA-rpplwta showed hydrophilic and have high stabilities in vitro and in vivo.3.¹⁸F-AlF-NOTA-atwlppr and ¹⁸F-AlF-NOTA-rpplwta micro PET/CT can visualize the NRP-1 positive（U-87MG）tumor clearly in vivo and have high tumor/brain ratios,which is useful for glioma imaging.Compare to ¹⁸F-AlF-NOTA-rpplwta,¹⁸F-AlF-NOTA-atwlppr has a higher tumor/liver ratio,suggesting that it is more suitable for whole body tumor imaging.