Preparation And Bioassay Of Positive Electron Labeling Nanoprobe ⁶⁸Ga-NOTA-DGL And ⁶⁸Ga-NOTA-DGL-PEG-RGDyC

ObjectiveSynthesize the NOTA-DGL and NOTA-DGL-PEG-RGDyC precursors and positron emission label them and biologically evaluate them in vitro and in vivo.Methods1.Synthesis of precursors.（1）Preparation of NOTA-DGL: DGL and NOTA-NHS were dissolved in DMSO solution at room temperature for 24 hours.After completion of the reaction,the aminoacetylation reaction was carried out and finally dialyzed with sodium acetate buffer.（2）Preparation of NOTA-DGL-PEG-RGDyC: NHS-PEG-MAL and RGDyC in the buffer solution of sodium acetate at room temperature for 5min,after the end of the reaction by adding DGL,boric acid buffer and then continue to respond 24 h After completion of the reaction,excess β-mercaptoethanol was added.NOTA-NHS was dissolved in DMSO solution and allowed to stand at room temperature for 24 h,and finally dialyzed against sodium acetate buffer.2.RadiolabelingThe ⁶⁸GaCl3 was leached from the germanium-gallium generator with hydrochloric acid and reacted with the precursor at 70 ° C for 10 to 15 minutes.3.Octanol-water partition coefficient4.In vitro stability studies5.Cell uptake and efflux studies6.Animal biodistribution studies（1）In vivo biological distribution of normal mice.（2）In vivo biological distribution of tumor-bearing mice.7.Micro PET imaging.8.Statistical analysis.Use SPSS 20.0 to analyze data.Data was expressed as mean ±SD where appropriate.P <0.05 was considered statistically significant.Results1.Characterization of the labeled precursors NOTA-DGL and NOTA-DGL-PEG-RGDyC.（1）The PEG and RGDyC were successfully connected to DGL by NMR spectroscopy to obtain DGL-PEG-RGDyC.（2）The NOTA of the labeled precursors NOTA-DGL and NOTA-DGL-PEG-RGDyC were successfully detected by UV absorbance test.2.Radiolabeling The imaging agent can be prepared in 15 mins.The radiochemical yield was between 50% 70%,and after separation and purification the radiochemical purity was more than 98%.PH is between 4.0 to 4.2,and the solution is colorless,transparent,clear with no other heavy metal pollution.3.Octanol-water partition coefficient LogP ⁶⁸Ga-NOTA-DGL =-1.02,LogP⁶⁸Ga-NOTA-DGL-PEG-RGDyC =-3.037,showed that the nanomolecular probe was water-soluble.4.In vitro stability studies.The two kinds of imaging agents were incubated in 0.1 M PBS and calf serum at 37 ℃ for 120 min after radiochemical purity> 95%,indicating good in vitro stability,specific activity reached 30 GBq / μmol.5.Cell uptake and efflux experiments.（1）cell uptake experiment.A549 and U87 MG cells could rapidly and efficiently uptake two imaging agents.The imaging agents further enhanced the uptook the two cell lines and reached the peak at 2 h after incubation.（2）cell efflux experiments.Both imaging agents showed a slow decrease with time in both cell lines,and there were some imaging agents retented at 2 h.6.Animal biodistribution studies.（1）In vivo biological distribution of normal mice.Imaging agents in the blood removed faster,radioactive-uptake was mainly in the liver and spleen,with other organs less radioactive-intake.（2）In vivo biological distribution of tumor-bearing mices.The radioactive-uptake in U87 MG tumor tissue were（0.19 ± 0.02）%ID / g and（0.27 ± 0.09）% ID / g respectively,lower than those of other major organ tissues after 1 hour injection of two imaging agents.7.Micro-PET imaging of tumor-bearing mice.After injection of two imaging agents through the tail vein of tumor-bearing mices,the imaging agents were mainly distributed in the liver,with tumor nodules less significant uptake.ConclusionsThe ⁶⁸Ga labeling of NOTA-DGL and NOTA-DGL-PEG-RGDyC proves that this method is simple,rapid and has high radiochemical yield.However,⁶⁸Ga-NOTA-DGL and ⁶⁸Ga-NOTA-DGL-PEG-RGDyC in tumor tissue uptake and imaging results are not ideal,need to be further improved and optimized.