Delta-Opioid Receptor (δOR) Targeted Near-Infrared Fluorescent Agent for Imaging of Lung Cancer: Synthesis and Evaluation In Vitro and In Vivo

Abstract

In the United States, lung cancer is the leading cause of cancer deaths and ranks second in the number of new cases annually among all types of cancers. Better methods or tools for diagnosing and treating this disease are needed to improve patient outcomes. The delta opioid receptor (δOR) is reported to be overexpressed in lung cancers and not expressed in normal lung. Thus, we decided to develop a lung cancer-specific imaging agent targeting this receptor. We have previously developed a δOR–targeted fluorescent imaging agent based on a synthetic peptide antagonist (Dmt-Tic) conjugated to a Cy5 fluorescent dye. In this work, we describe the synthesis of Dmt-Tic conjugated to a longer wavelength near-infrared fluorescent (NIRF) dye, Li-cor IR800CW. Binding affinity of Dmt-Tic-IR800 for the δOR was studied using lanthanide time-resolved fluorescence (LTRF) competitive binding assays in cells engineered to overexpress the δOR. In addition, we identified lung cancer cell lines with high- and low-endogenous expression of the δOR. We confirmed protein expression in these cell lines using confocal fluorescence microscopy imaging and used this technique to estimate the cell-surface receptor number in the endogenously expressing lung cancer cell lines. The selectivity of Dmt-Tic-IR800 for imaging of the δOR in vivo was shown using both engineered cell lines and endogenously expressing lung cancer cells in subcutaneous xenograft models in mice. In conclusion, the δOR–specific fluorescent probe developed in this study displays excellent potential for imaging of lung cancer.

TricarbocyanineN-triazoles: the scaffold-of-choice for long-term near-infrared imaging of immune cellsin vivo

“…800–900 nm). We compared the fluorescence staining of CIR38M to the commercially available IR800CW-SE , a gold standard in NIR fluorescence labelling, 46 , 47 by treating CD4 + T cells with both fluorophores under the same conditions and visualising them under a fluorescence microscope. Counter-staining with the green fluorophore CellTracker Green was used to confirm CD4 + T cell labelling.…”

Investigating endogenous µ-opioid receptors in human keratinocytes as pharmacological targets using novel fluorescent ligand

“…This has inspired to develop fluorophore-conjugated OR ligands to study endogenous cellular trafficking and opioid ligand-receptor dynamics in skin cells under different conditions. Previous reports have shown that fluorophore-conjugated OR ligands (peptide and non-peptide) can detect receptors at low density and allowed analysis of the fast kinetics of receptor ligand-interaction and flow cytometric sorting of OR-containing cell populations [ 27 – 33 ]. We chose Endomorphine-1 (EM), a high affinity endogenous ligand for the μ-OR, conjugated to Tetramethylrhodamine (TAMRA) to examine the cell biology of the μ-OR in keratinocytes [ 34 ].…”

The delta opioid receptor tool box

“…Nevertheless, a wide range of fluorescent conjugates of delta agonists and antagonists have been synthesized and are commercially available (Cell Aura Technologies Ltd. and Cisbio Bioassays). Fluorescent antagonists have proved useful to examine delta opioid receptor distribution in living cells (Kshirsagar et al, 1998; Balboni et al, 2004; Cohen et al, 2016); while agonists have been used to study delta opioid receptor inter-nalization and trafficking in vivo (Arttamangkul et al, 2000; Cahill et al, 2001; Lee et al, 2002). …”