Preparation And Properties Of Novel PET Probe For Furin-targeted Tumor Imaging

Early diagnosis of tumor is of great importance for human being to fight tumors. Among the techniques for tumor diagnosis, Positron Emission Tomography（PET） has been widely used due to its advantages in early diagnosis of tumor with high sensitivity and accuracy. Therefore, development of novel PET probes is vital for broadening the clinical applications of PET. To achieve a successful tumor imaging, design biocompatible probes for specific target can overcome the delivery barriers to tumors with high specificity. The target of this paper is furin. Furin is a cellular endoprotease which is upregulated in several cancers. Over-expression of furin increases the invasiveness and growth of tumor. So design PET probes for furin will be useful for early diagnosis of tumor.In this study, novel PET probe ¹⁸F-FBCKR targeting furin was designed and the furin-associated enzymatic self-assembly with PET imaging was firstly integrated. In detail, based on a new condensation reaction, a F-18 PET probe with furin cleavage substrates was designed. The probe,¹⁸F-FBCKR was designed to have these components as following:（1） a RVRR peptide sequence for cell membrane translocation and furin cleavage;（2） a disulfided Cys for supplying the 1,2-aminothiol group for condensation with the cyano group on the 2-cyanobenzothiazole（CBT） motif;（3） a Lys between Cys and CBT whose side chain ε-NH2 was covalently conjugated with ¹⁸F-FBA for PET imaging. After being uptaken by tumor cells overexpressing furin, the probes will be hydrolyzed by furin to initiate the condensation reaction, thereafter self-assemble into radioactive nanoparticles in tumor cells and can enhance the radioactivity for tumor-targeted PET imaging. Nanomaterials as PET imaging agents are widely explored in nowadays due to their high surface areas（or surface/volume ratio）, which could easily enhanced the concentration of imaging agents in region of interest. Therefore, these radioactive nanoparticles self-assembled by the new PET probe in this paper are highly “smart” and tumor-targeted which are totally different from those premade nanoparticles with synthetic difficulty, low uptake and targeting deficiency in tumor, etc.Firstly, the precursor CKR and the cold analog FBCKR were synthesized. The syntheses were facile. After purification with HPLC, the products were characterized with NMR and MS.To validate furin-controlled condensation and self-assembly, FBCKR was used for the nano preparation in vitro. After incubation of FBCKR at 37°C with furin, the incubation mixture was directly injected into a HPLC system and the peaks were collected for MALDI-MS analysis. Peaks on HPLC traces were identified as the condensation products of FBCKR after furin cleavage（Dimer）. Time course HPLC analysis of furin cleavage indicated that, at this condition, furin-controlled condensation of FBCKR was accomplished in 2h and was stable until 8 h. Directly taking the above dispersion for SEM and TEM observation, we found that the nanoparticles had an average diameter of 207 ± 99 nm under SEM and 182±70 nm under TEM.Radiosynthesis of ¹⁸F-FBCKR was conducted. ¹⁸F-SFB was synthesized automatically using multifunctional synthesizer. Labeling of precursor CKR with ¹⁸F-SFB yielded ¹⁸F-FBCKR after HPLC purification. Optimization of synthesis conditions of ¹⁸F-FBCKR were conducted, and the best p H was 7.2, reaction temperature was 50 °C and reaction time was 0.5h. Quality control showed that ¹⁸F-FBCKR met the requirements for injection; stability study indicated that ¹⁸F-FBCKR was stable in saline and fetal bovine serum at 37 °C up to 5 h.The cell permeability of ¹⁸F-FBCKR and FBCKR for the cellular experiments was studied. The expression level of furin in MDA-MB-468 was tested with western blot and Lo Vo cells were chosen as control. The results indicated that MDA-MB-468 cells revealed a positive signal for furin（31.2% of β-actin） while very weak signal of furin（10.4% of β-actin） was detected in Lo Vo cells. Thus MDA-MB-468 cells were chosen for cellular experiments. Then the biocompatibility of FBCKR was studied. MTT assay indicated FBCKR was safe for the co-injection with ¹⁸F-FBCKR. Then fluorescence imaging of the cells was conducted. For the MDA-MB-468 cells incubated with FBCKR at 37 °C, strong fluorescence emissions inside the cells were clearly observed, and the condensation products of FBCKR were actually at or near the localizations of furin（i.e., Golgi bodies）, also nanoparticles were directly observed with power electron microscope. Cell permeability study of ¹⁸F-FBCKR was conducted by incubating the probes with MDA-MB-468 cells and the cellular uptake rapidly reached its maximum of 22.2% at 60 min, suggesting the MDA-MB-468 cells uptake ¹⁸F-FBCKR quickly and efficiently. Cellular efflux titration was also conducted. The results showed that 120 min after cell efflux, cells only incubated with ¹⁸F-FBCKR had 13.1 ± 1.2% of initial radioactivity retained while those cells co-incubated with 5, 25, 50 μM FBCKR had 20.3 ± 4.2%, 28.5 ± 1.4%, 32.1 ± 0.9% of initial radioactivity retained, respectively. The results indicated that co-injection of FBCKR with ¹⁸F-FBCKR artificially saturated the Cys in cancer cells and ensure the feasibility of intracellular self-assembly NPs.Then small animal imaging PET scans was done using MDA-MB-468 tumor-bearing nude mice each co-injected with ¹⁸F-FBCKR and FBCKR（groupⅡ） via tail vein. Those nude mice only injected with same dosage of ¹⁸F-FBCKR（groupⅠ） were studied in parallel. Micro PET imaging results showed that tumors in those mice that only injected with ¹⁸F-FBCKR could be visualized at 30 min and 60 min post injection but their PET images attenuated very fast after 60 min. But tumors on the mice co-injected with ¹⁸F-FBCKR and FBCKR were clearly visualized with good tumor-to-background contrast from 30 to 360 min. Quantitative results demonstrated that tumor uptake ratios between groupⅡand groupⅠare 2.5（at 10 min）, 3.6（at 30 min）, 6.4（at 60 min）, 7.9（at 120 min）, 8.2（at 240 min） and 6.5（at 360 min）, respectively. These results suggested that the radioactive probe ¹⁸F-FBCKR had a good in vivo tumor uptake enough for tumor PET imaging but co-injection of ¹⁸F-FBCKR with FBCKR resulted in better tumor uptake and longer attenuation of the radioactivity in the tumor, indicating that FBCKR helped the furin-controlled intracellular condensation and self-assembly of NPs in tumors.These properties show that the new compound ¹⁸F-FBCKR is a novel promising PET probe.