The study for characterization and integrinαVβ3 targeting of 64Cu-cRGDyK-HSA

Abstract

Introduction: RGD is famous integrinαVβ3 targeting peptide, so integrinαVβ3 targeting for tumor imaging is useful for tumor imaging. However, RGD has the short circulation time and the majority of the injected probes are cleared through the renal system or hepatobiliary system. To enhance the half-lives of RGD and tumor targeting, cRGDyK was conjugated to HSA via bioorthogonal click reaction. To develop the best RGD conjugated HSA nanoparticles, two types of cRGDyK conjugated HSA were synthesized and the probes (cRGDyK-HSA) were characterized, radiolabeled and preliminarily tested in in vitro and in vivo properties of integrinαVβ3 expressing cancer targeting. Methods: HSA was conjugated with DBCO-NHS ester (molar ratio of HSA : DBCO-NHS ester

1 : 5.62 for reaction 1 and 1 : 11.24 for reaction 2) for click reaction linker. And DBCO-HSA was conjugated with N3-cRGDyK (HSA-DBCO : N3-cRGDyK

1 : 3 for reaction 1 and 1 : 6 for reaction 2). For radiolabeling, 64Cu-labeled 3-azidopropyl-NOTA was conjugated to DBCO-HSA and cRGDyK-HSA via click reaction. At each conjugation step, the conjugates were purified using PD-10 column, eluted with the PBS buffer. All conjugation products were analyzed via MALDI-TOF-MS and radiolabeling efficiencies were measured by instant thin layer chromatography (ITLC). The stability of 64Cu-labeled HSA and -cRGDyK-HSA in serum were monitored during 48 hrs. To certify cRGDyK-HSA binding to integrinαVβ3 in cell level, FNR648-labeled cRGDyK-HSA was used for confocal microscopy imaging. 64Cu-labeled HSA and -cRGDyK-HSA were intravenously injected to SK-OV3 tumor bearing mice and the distribution of the probes in mice were imaged by small animal PET at 10 min, 4 hours, 24 hours and 48 hours post injection (p.i.).. Results: DBCO-NHS ester and cRGDyK were successfully conjugated to HSA, according to their molar ratio. In case of DBCO, the number of DBCO conjugated to HSA

reaction 1 was 3.94 ± 0.70 and reaction 2 was 6.72 ± 0.41. In case of cRGDyK, the number of cRGDyK conjugated to DBCO-HSA

reaction 1 was 2.07 ± 0.51 and reaction 2 was 5.29 ± 0.76. Radiolabeling efficiencies of 64Cu-HSA and 64Cu-cRGDyK-HSA (reaction 2) was 100% and after PD-10 purification, that of 64Cu-cRGDyK-HSA (reaction 1) was almost 100%. 64Cu-HSA and -cRGDyK-HSA were stable in serum after 48 hours incubation. Confocal microscopy images showed that FNR648-labeled cRGDyK-HSA were localized in cell membrane and intracellular regions, this localization in cells was blocked when cells were pre-incubated with excess cRGDyK. The cell uptake of 64Cu-labeled cRGDyK-HSA (reaction 1 and reaction 2) was higher in SK-OV3 cells (integrinαVβ3 positive) than 22Rv1 cells (integrinαVβ3 negative, P < 0.05). PET images revealed that reaction 2 of cRGDyK-conjugated HSA had the highest uptake in tumor (5.37 ± 1.09 %ID/g). Conclusion: DBCO-HSA and cRGDyK-HSA were successfully synthesized. As the molar ratio of DBCO or cRGDyK were different, the number of attached DBCO or cRGDyK to HSA were consistently different. Using click reaction, 64Cu was successfully labeled to HSA and cRGDyK-HSA. cRGDyK-HSA could bind integrinαVβ3 in tumor cells and in vivo PET imaging results probed that the 64Cu-cRGDyK-HSA could target tumor after 4 hours p.i.. These results demonstrate that 64Cu-labeled cRGDyK-HSA can be used as PET tumor imaging probes.