In Vitro and In Vivo Evaluation of Targeted Fluorescent Imaging Agents for Diagnosis and Resection of Cancer.

Abstract

Local re-occurrence of cancer in patients with solid tumors is currently the most common reason for failure of treatment strategies. This fact indicates that prevailing approaches for tumor resection can cure only 50% of patients. A major cause of failure in tumor resection is off-target drug cytotoxicity and lack of sensitivity in tumor detection methods. These disadvantages are addressed with the development of targeted therapy and diagnostics, which significantly aid treatment strategies. Targeted diagnostics exploit properties of tumor cells that show significant up-regulation of tumor biomarkers. These biomarkers are targeted by a homing ligand attached to a fluorophore for visual inspection during surgery. However, these approaches suffer from disadvantages like high autofluorescence from background tissues, tissue absorption, and scattering, resulting in decreased image sensitivity and resolution. The use of near-infrared (NIR) fluorophores to overcome these drawbacks has generated unprecedented interest among researchers. The NIR window lies within the range of 650 to 1,700 nm, which results in reduced absorption and scattering by the tissues, thereby providing deeper tissue penetration and reduced autofluorescence. NIR fluorophores can be designed to target tumor biomarkers such as prostate specific membrane antigen (PSMA) or folate receptors found over-expressed on cancer tissues. These targeted fluorophores consist of small-molecule ligands conjugated with NIR dyes that bind with high specificity to PSMA and folic acid receptors. In this protocol, we have extensively described the methodology for the synthesis of targeted NIR agents for PSMA (DUPA-NIR bioconjugate) and folic acid (folate-NIR bioconjugate), along with detailed steps for preclinical evaluation. Procedures to calculate the binding affinity to cancer cells in vitro are described, along with uptake and biodistribution in different mice models in vivo. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and purification of DUPA and folate-peptide linkers via a SPPS strategy Basic Protocol 2: Conjugation, purification, and characterization of targeted bioconjugates with NIR probe for deep-tissue imaging applications Basic Protocol 3: In vitro evaluation of binding affinity of targeted DUPA-NIR and folate-NIR bioconjugates using a spectrophotometer Basic Protocol 4: Induction of tumor in mice to develop CDX or metastatic tumor models Basic Protocol 5: Intravenous administration of targeted DUPA-NIR and folate-NIR bioconjugates in mouse CDX or metastatic tumor models for deep-tissue NIR imaging and tumor resection.