Near-Infrared Fluorescent RGD Peptides for Optical Imaging of Integrin α_vβ₃ Expression in Living Mice

Abstract

Near-infrared fluorescence optical imaging is a powerful technique for studying diseases at the molecular level in preclinical models. We recently reported that monomeric RGD peptide c(RGDyK) conjugated to the NIR fluorescent dye specifically targets integrin receptor both in cell culture and in living subjects. In this report, Cy5.5-conjugated mono-, di-, and tetrameric RGD peptides were evaluated in a subcutaneous U87MG glioblastoma xenograft model in order to investigate the effect of multimerization of RGD peptide on integrin avidity and tumor targeting efficacy. The binding affinities of Cy5.5-conjugated RGD monomer, dimer, and tetramer for α v β 3 integrin expressed on U87MG cell surface were determined to be 42.9 ± 1.2, 27.5 ± 1.2, and 12.1 ± 1.3 nmol/L, respectively. All three peptide-dye conjugates had integrin specific uptake both in vitro and in vivo. The subcutaneous U87MG tumor can be clearly visualized with each of these three fluorescent probes. Among them, tetramer displayed highest tumor uptake and tumor-tonormal tissue ratio from 0.5 to 4 h postinjection. Tumor-to-normal tissue ratio for Cy5.5-conjugated RGD monomer, dimer, and tetramer were found to be 3.18 ± 0.16, 2.98 ± 0.05, and 3.63 ± 0.09, respectively, at 4 h postinjection. These results suggest that Cy5.5-conjugated monomeric, dimeric, and tetrameric RGD peptides are all suitable for integrin expression imaging. The multmerization of RGD peptide results in moderate improvement of imaging characteristics of the tetramer, compared to that of the monomer and dimeric counterparts.

Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma

“…In vivo contrast is measured as the ratio of signal in mice with tumors to signal in mice without tumors (sham surgery controls). IC 50 (40)(41)(42), but attempts to image glioblastoma cells injected into the mouse forebrain were unsuccessful (40). Removal of the skull before imaging revealed only minimal contrast between tumor cells and normal brain (43).…”

“…45 and c(RGDfK) in Table 1]. Many RGD-containing molecules have required multimerization and the attachment of carbohydrates or synthetic polymers to improve tumor targeting, pharmacokinetic profiles, and tissue distribution (42,45). Although such modifications could potentially further enhance the tumor imaging contrast observed with EETI 2.5F, they make manufacturing and clinical development more expensive and challenging.…”

Real-Time Intravital Imaging of RGD−Quantum Dot Binding to Luminal Endothelium in Mouse Tumor Neovasculature

“…To confirm this finding, in our subsequent control we blocked integrin α v β 3 binding sites by presaturating with an RGD-Cy5.5 conjugate which was previously validated to bind to α v β 3 in cell culture and in living mice. 30 Due to extravasation and consequent blurring of the vessel Table 1). More common were instances in which RGD-qdots appeared to gently rock, roll, and temporarily stop as they moved across the neovasculature endothelial lining (movie S4 in Supporting Information).…”

“…4,5,[29][30][31] Nonspecific binding in tumors could nevertheless be caused either by nonspecific adsorption of the RGD peptide or by the qdot surface to tumor neovasculature. To control for this, similar peptide RAD was employed on qdots.…”

“…To confirm this finding, in our subsequent control we blocked integrin α v β 3 binding sites by presaturating with an RGD-Cy5.5 conjugate which was previously validated to bind to α v β 3 in cell culture and in living mice. 30 Due to extravasation and consequent blurring of the vessel edges, binding of RGD-Cy5.5 to tumor vessel walls was not observable (see red channel in movie 4 in Supporting Information). Administration of RGD-qdots (50 pmol) shortly after blocking peptide led to only rare binding of the conjugate to vessels (see Figures 3 and 4 and Table 1).…”