Abstract
The detection of human malignancies by near-infrared (NIR) fluorescence will require the conjugation of cancer-specific ligands to NIR fluorophores that have optimal photoproperties and pharmacokinetics. IRDye78, a tetra-sulfonated heptamethine indocyanine NIR fluorophore, meets most of the criteria for an in vivo imaging agent, and is available as an N-hydroxysuccinimide ester for conjugation to low-molecular-weight ligands. However, IRDye78 has a high charge-to-mass ratio, complicating purification of conjugates. It also has a potentially labile linkage between fluorophore and ligand. We have developed an ion-pairing purification strategy for IRDye78 that can be performed with a standard C18 column under neutral conditions, thus preserving the stability of fluorophore, ligand, and conjugate. By employing parallel evaporative light scatter and absorbance detectors, all reactants and products are identified, and conjugate purity is maximized. We describe reversible and irreversible conversions of IRDye78 that can occur during sample purification, and describe methods for preserving conjugate stability. Using seven ligands, spanning several classes of small molecules and peptides (neutral, charged, and/or hydrophobic), we illustrate the robustness of these methods, and confirm that IRDye78 conjugates so purified retain bioactivity and permit NIR fluorescence imaging of specific targets.
Highlights
The detection of cancer cells and their products in vivo is dependent on both the signal generated by the contrast agent and the inherent background or ‘‘noise’’ of the tissue being imaged
When the conjugation reaction was performed in aqueous buffer, nucleophilic attack by the hydroxyl ion of H2O can occur, resulting in cleavage of the N-hydroxysuccinimide ester (NHS) ester bond and formation of the carboxylic acid form of IRDye78 (IRDye78-CA; not shown)
For NIR fluorescence imaging of cancer cells to become a clinical reality, convenient, reliable, and generally applicable methods are needed for producing stable fluorophore/ligand conjugates in high yield and with high purity
Summary
The detection of cancer cells and their products in vivo is dependent on both the signal generated by the contrast agent and the inherent background or ‘‘noise’’ of the tissue being imaged. The technique of nearinfrared (NIR) fluorescence imaging provides extremely low background since living tissue has minimal absorption and autofluorescence in the NIR wavelength range of 700 –900 nm, especially when compared to visible (400– 700 nm) light [1]. By conjugating a cancer-specific ligand to such an NIR fluorophore, a novel contrast agent is generated that has the potential for highly specific and sensitive detection of human malignancy. Indocyanines have a long history of clinical use. In 1956, indocyanine green (ICG; IC-GREEN2) was FDA-approved for use in indicatordilution studies in humans and has had a remarkably good safety profile
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