RESUMO
In recent years, significant progress has been made in the development of fluorescent contrast agents for clinical applications. For the development of a fluorescent probe, it is crucial to evaluate its safety profile, including biodistribution. Specific methods need to be developed for the absolute quantification of fluorescent probes in tissue specimens from animals administered with test compounds in the framework of biodistribution/efficacy/toxicity studies. Here, we describe a new method for the absolute quantification of fluorescent probes in tissue specimens from animals administered with compounds that have absorption and emission wavelength in the Near-Infrared region (600-800 nm). The protocol is based on the standard addition approach in order to minimize the interference of the matrix on the analyte signal causing inaccuracy in the absolute determination of the concentration. The measurement of the fluorescence intensity is done via a microplate reader. The method has been fully validated and applied for the quantification of a fluorescence-guided surgery targeted contrast agent in a Good Laboratory Practice (GLP) biodistribution study. Results clearly demonstrate that this procedure is fully applicable in a preclinical setting and that it overcomes common issues associated with fluorescence signal quantification in tissue extracts.
Assuntos
Corantes Fluorescentes , Extratos de Tecidos , Animais , Fluorescência , Distribuição TecidualRESUMO
Polydentate chelators are an important part of an imaging probe, which consists of an agent that usually produces signals for imaging purposes connected to a targeting moiety. The goal of this study was to set up a generic protocol to prepare a library of polydentate ligands having a 6-amino-6-methyl-1,4-perhydrodiazepine (AMPED) core and able to chelate metal ions of interest for various diagnostic imaging techniques, including Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT). These ions, among which we can include Mn(ii), Cu(ii), Al(iii) or Ga(iii), require penta- or hexa-dentate chelators for this purpose, and the AMPED scaffold has considerable potential to support various pendant arms for coordination of such ions. AMPED already has three amino nitrogen donors; thus, only two or three additional arms should be introduced to obtain penta- or hexa-dentate systems. This condition implies that symmetrical or asymmetrical structures have to be developed, depending on the functionalization of cyclic and exocyclic amines. Starting from easily available materials, we have designed a convenient protocol for the preparation of multiple AMPED-based ligands endowed with different characteristics, several of which were synthesized as examples.