Preparation, In Vitro Affinity, and In Vivo Biodistribution of Receptor-Specific 68Ga-Labeled Peptides Targeting Vascular Endothelial Growth Factor Receptors

Bioconjug Chem. 2022 Oct 19;33(10):1825-1836. doi: 10.1021/acs.bioconjchem.2c00272. Epub 2022 Oct 5.


As angiogenesis plays a key role in tumor growth and metastasis, the angiogenic process has attracted scientific interest as a target for diagnostic and therapeutic agents. Factors influencing angiogenesis include the vascular endothelial growth factor (VEGF) family and the two associated receptor types (VEGFR-1 and VEGFR-2). VEGFR-1/-2 detection and quantification in cancer lesions are essential for tumor process management. As a result of the advantageous pharmacokinetics and image contrast, peptides radiolabeled with PET emitters have become interesting tools for the visualization of VEGFR-1/-2-positive tumors. In this study, we prepared 68Ga-labeled peptides containing 15 (peptide 1) and 23 (peptide 2) amino acids as new PET tracers for tumor angiogenic process imaging.

Methods: The peptides were conjugated with NODAGA-tris(t-Bu ester) and subsequently radiolabeled with [68Ga]Ga-chloride. The prepared [68Ga]Ga-NODAGA-peptide 1 and [68Ga]Ga-NODAGA-peptide 2 were tested for radiochemical purity and saline/plasma stability. Consequently, the binding affinity toward VEGFRs was assessed in vitro on human glioblastoma and kidney carcinoma cells. The found peptide receptor affinity was compared with the calculated values in the PROtein binDIng enerGY prediction (PRODIGY) server. Finally, the biodistribution study was performed on BALB/c female mice to reveal the basic pharmacokinetic behavior of radiopeptides.

Results: The in vitro affinity testing of [68Ga]Ga-NODAGA-peptides 1 and 2 showed retained receptor binding as characterized by equilibrium dissociation constant (KD) values in the range of 0.5-1.2 μM and inhibitory concentration 50% (IC50) values in the range of 3.0-5.6 μM. Better binding properties of peptide 2 to VEGFR-1/-2 were found in the PRODIGY server. The biodistribution study on mice showed remarkable accumulation of both peptides in the kidneys and urinary bladder with a short half-life after intravenous application. The in vitro plasma stability of [68Ga]Ga-NODAGA-peptide 2 was superior to that of [68Ga]Ga-NODAGA-peptide 1.

Conclusions: The obtained results demonstrated a high radiolabeling yield with no need for purification and preserved binding potency of 68Ga-labeled peptides 1 and 2 toward VEGFRs in cancer cells. The peptide-receptor protein interaction assessed in protein-peptide docking determined the strongest interaction of peptide 2 with domain 2 of VEGFR-2 in addition to a more acceptable plasma stability (t1/2 = 120 min) than that for peptide 1. We found both radiolabeled peptides very potent in their receptor binding, which makes them suitable imaging agents. The rapid transition of the radiopeptides into the urinary tract indicates suitable pharmacokinetic characteristics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids
  • Animals
  • Cell Line, Tumor
  • Chlorides
  • Esters
  • Female
  • Gallium Radioisotopes* / chemistry
  • Heterocyclic Compounds, 1-Ring / chemistry
  • Humans
  • Mice
  • Mice, Inbred BALB C
  • Peptides / chemistry
  • Positron-Emission Tomography / methods
  • Receptors, Vascular Endothelial Growth Factor
  • Tissue Distribution
  • Vascular Endothelial Growth Factor A*
  • Vascular Endothelial Growth Factor Receptor-1
  • Vascular Endothelial Growth Factor Receptor-2


  • 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane
  • Gallium Radioisotopes
  • Vascular Endothelial Growth Factor A
  • Vascular Endothelial Growth Factor Receptor-2
  • Vascular Endothelial Growth Factor Receptor-1
  • Chlorides
  • Heterocyclic Compounds, 1-Ring
  • Peptides
  • Receptors, Vascular Endothelial Growth Factor
  • Amino Acids
  • Esters