Arg–Arg–Leu
(RRL) is a potent tumor-homing tripeptide.
However, the binding target is unclear. In this study, we intended
to identify the binding target of RRL and evaluate the tumor targeting
of 99mTc-MAG3-RRL in vivo.
Biotin–RRL, 5-TAMRA-RRL, and 99mTc-MAG3-RRL were designed to trace the binding target and tumor lesion.
Immunoprecipitation-mass spectrometry was conducted to identify the
candidate proteins and determination of the subcellular localization
was also performed. A pull-down assay was performed to demonstrate
the immunoprecipitate. Fluorescence colocalization and cell uptake
assays were performed to elucidate the correlation between the selected
binding protein and RRL, and the internalization mechanism of RRL.
Biodistribution and in vivo imaging were performed
to evaluate the tumor accumulation and targeting of 99mTc-MAG3-RRL. The target for RRL was screened to be heat
shock protein 70 (HSP70). The prominent uptake distribution of RRL
was concentrated in the membrane and cytoplasm. A pull-down assay
demonstrated the existence of HSP70 in the biotin–RRL captured
complex. Regarding fluorescence colocalization and cell uptake assays,
RRL may interact with HSP70 at the nucleotide-binding domain (NBD).
Clathrin-dependent endocytosis and macropinocytosis could be a vital
internalization mechanism of RRL. In vivo imaging
and biodistribution both demonstrated that 99mTc-MAG3-RRL can trace tumors with satisfactory accumulation in hepatoma
xenograft mice. The radioactive signals accumulated in tumor lesions
can be blocked by VER-155008, which can bind to the NBD of HSP70.
Our findings revealed that RRL may interact with HSP70 and that 99mTc-MAG3-RRL could be a prospective probe for
visualizing overexpressed HSP70 tumor sections.