Synthesis, Chemical Characterization and Multiscale Biological Evaluation of a Dimeric-cRGD Peptide for Targeted Imaging of α V β 3 Integrin Activity

Abstract

Cyclic peptides containing the Arg-Gly-Asp (RGD) sequence have been shown to specifically bind the angiogenesis biomarker α _V β ₃ integrin. We report the synthesis, chemical characterization, and biological evaluation of two novel dimeric cyclic RGD-based molecular probes for the targeted imaging of α _V β ₃ activity (a radiolabeled version, ⁶⁴Cu-NOTA-PEG₄-cRGD₂, for PET imaging, and a fluorescent version, FITC-PEG₄-cRGD₂, for in vitro work). We investigated the performance of this probe at the receptor, cell, organ, and whole-body levels, including its use to detect diabetes associated impairment of ischemia-induced myocardial angiogenesis. Both versions of the probe were found to be stable, demonstrated fast receptor association constants, and showed high specificity for α _V β ₃ in HUVECs (K _d ~ 35 nM). Dynamic PET-CT imaging indicated rapid blood clearance via kidney filtration, and accumulation within α _V β ₃-positive infarcted myocardium. ⁶⁴Cu-NOTA-PEG₄-cRGD₂ demonstrated a favorable biodistribution, slow washout, and excellent performance with respect to the quality of the PET-CT images obtained. Importantly, the ratio of probe uptake in infarcted heart tissue compared to normal tissue was significantly higher in non-diabetic rats than in diabetic ones. Overall, our probes are promising agents for non-invasive quantitative imaging of α _V β ₃ expression, both in vitro and in vivo.

Figure 1

Chemical structures of (A) NOTA-PEG₄-cRGD₂ and (B) FITC-PEG₄-cRGD₂.

Figure 2

SPR sensograms depicting binding between cRGD ligands and α _V β ₃ integrin receptor. BIAcore 3000 kinetics studies of interactions between immobilized integrin α _V β ₃ receptor and (A) monomeric cyclic RGD probe (NOTA-cRGD), (B) dimeric cyclic RGD (NOTA-PEG₄-cRGD₂) probe, (C) cRGD₂ conjugated with FITC (FITC-PEG₄-cRGD₂), and (D) NOTA-PEG₄-cRGD₂ labeled with non-radioactive Cu²⁺. Kinetic studies were performed at a 30 μ L/min flow rate, with a 4 min association followed by a 10 min dissociation period.

Figure 3

Colocalization between phycoerythrin-labeled anti- α _V β ₃ integrin primary antibody (PE-LM609) and FITC-labeled PEG₄-cRGD₂ probe. Human umbilical vein endothelial cells (HUVEC) were grown to confluency and incubated with both PE-LM609 and FITC-PEG₄-cRGD₂. Fluorescence microscopy images were acquired in DAPI/PE (A) and DAPI/FITC (B) channels and were superimposed to create colocalized pixel map (C) to calculate Pearson’s coefficient. Flow cytometric analysis of HUVEC co-incubated with PE-LM609 and FITC-PEG₄-cRGD₂ demonstrated a very high degree of colocalization between integrin α _V β ₃ and FITC-PEG₄-cRGD₂ probe (D).

Figure 4

Binding kinetics of FITC and ⁶⁴Cu labeled cRGD ₂ probes. (A) Radioactivity of confluent HUVEC cells incubated with varying concentrations of ⁶⁴Cu-NOTA-PEG₄-cRGD₂ (green) and with either 20 μM EDTA (red) or 50 μM of H-PEG₄-cRGD₂ (blue). (B) Fluorescence of the FITC-PEG₄-cRGD₂ (green) with either 20 μM EDTA (red) or 50 μM of H-PEG₄-cRGD₂ (blue). (C) Comparison of the fluorescence of the FITC-PEG₄-cRGD₂ (green) with FITC-Galacto-cRGD₂ (orange). (D) Correlation between FITC and Cu⁶⁴ labeled cRGD₂ probes bound to HUVECs. (E) The fluorescence of HUVEC cells incubated with varying concentrations of FITC-PEG₄-cRGD₂ (green) and co-incubated with 20 μM of Mn²⁺ (purple). (F) Radioactivity of confluent HUVEC cells incubated with varying concentrations of ⁶⁴Cu-NOTA-PEG₄-cRGD₂ (green) and co-incubated with 20 μM of Mn²⁺ (purple). (G) Competition binding between ⁶⁴Cu-NOTA-PEG₄-cRGD₂ (50 nM) and increasing concentrations of unlabeled H-PEG₄-cRGD₂ in HUVEC cells.

Figure 5

Dynamic PET-CT images (A) were used to plot the blood clearance (B) and time activity curves (TAC) of ⁶⁴Cu-NOTA-PEG₄-cRGD₂ in selected organs (C). (D) Biodistribution of ⁶⁴Cu-NOTA-PEG₄-cRGD₂ in selected organs at 90 min post-injection in Lewis rats subjected to myocardial infarction induced by surgical ligation of LAD. Results are expressed in percentage of injected dose per gram tissue (%I.D./g). These results suggest a rapid blood clearance through renal filtration and very low non-specific uptake in other critical organs.

Figure 6

Representative in vivo hybrid PET-CT reconstructed short-axis (SA), vertical- (VLA) and horizontal long-axis (HLA) images acquired with iodinated contrast agent (Omnipaque) at 90 min post-injection of ⁶⁴Cu-NOTA-PEG₄-cRGD₂ (A). The iodinated blood pool contrast agent permitted better definition of right (RV) and left ventricle (LV) within the myocardium which is contoured with solid white line. Focal uptake of ⁶⁴Cu-NOTA-PEG₄-cRGD₂ was seen within anteriolateral LV regions (dashed yellow arrow) although significant uptake was seen in chest wall (CH) at the thoracotomy site (solid yellow arrows) indicating active wound healing associated α _V β ₃ receptor expression. Bull’s eye myocardial plots of ⁶⁴Cu-NOTA-PEG₄-cRGD₂ activity in diabetic and non-diabetic Lewis rats subjected to surgical ligation of LAD to induce myocardial infarction (arrow) (B). Hearts were immediately excised at 120 min post-injection, cleaned and filled with inert dental molding material and cut in four 2-mm thick slices from apex to base. After removing right ventricle (RV), each left ventricular (LV) slice was cut in four segments (anterior, septal, posterior, lateral), and ⁶⁴Cu radioactivity was measured in each segment with gamma well counting. Data for each slice were expressed as percentage of injected dose per gram tissue (%I.D./g) and categorized as infarct, border and remote areas (C).

Figure 7

α _V β ₃ immunohistochemistry. (A) A representative H&E stain of the infarct and remote areas of the heart. (B–E) representative immunohistochemistry images with LM609 (red) and DAPI (blue). (F) Quantification of α _V β ₃ positive area in stained heart tissue. The non-DM infract tissue showed significant increase in α _V β ₃ expression relative to DM infract tissue.