IRDye78 conjugates for near-infrared fluorescence imaging

Area of Science:

• Biomedical Imaging
• Organic Chemistry
• Analytical Chemistry

Background:

• Near-infrared (NIR) fluorescence imaging requires specific ligands conjugated to NIR fluorophores for detecting human malignancies.
• IRDye78 is a suitable NIR fluorophore but presents purification challenges due to its high charge-to-mass ratio and potentially labile linkage.

Purpose of the Study:

• To develop an effective purification strategy for IRDye78 conjugates.
• To ensure the stability and bioactivity of fluorophore-ligand conjugates for in vivo imaging.

Main Methods:

• An ion-pairing purification strategy using a standard C18 column under neutral conditions was developed.
• Parallel evaporative light scatter and absorbance detectors were employed to identify reactants and products, maximizing conjugate purity.
• Methods for preserving conjugate stability during purification were established.

Main Results:

• The ion-pairing strategy successfully purified IRDye78 conjugates under neutral conditions, preserving stability.
• The method identified and mitigated reversible and irreversible conversions of IRDye78 during purification.
• Purified conjugates using seven diverse ligands retained bioactivity and enabled NIR fluorescence imaging of specific targets.

Conclusions:

• A robust and versatile ion-pairing purification method for IRDye78 conjugates has been established.
• This method overcomes previous purification limitations, enhancing the utility of IRDye78 for in vivo cancer imaging.
• The developed strategy ensures the integrity and functionality of NIR fluorophore conjugates for biomedical applications.