Fluorescence lifetime imaging of activatable target specific molecular probes

Area of Science:

• Biomedical Optics
• Molecular Imaging
• Cell Biology

Background:

• Optical imaging with activatable antibody-fluorophore conjugates offers high target-to-background ratios.
• These probes are designed to become fluorescent upon cellular binding and internalization.
• Fluorescence lifetime imaging (FLI) is a potential method to monitor probe activation and internalization.

Purpose of the Study:

• To investigate the utility of fluorescence lifetime imaging (FLI) for monitoring cellular internalization of activatable antibody-fluorophore conjugates.
• To synthesize and characterize antibody-fluorophore conjugates for HER2/neu targeting.

Main Methods:

• Synthesis of trastuzumab-Alexa Fluor750 conjugates at 1:8 and 1:1 ratios.
• Measurement of fluorescence intensity and lifetime in vitro, including after dequenching with SDS.
• In vitro imaging experiments using HER2-positive (3T3/HER2(+)) and HER2-negative (BALB/3T3) cell lines.
• In vivo imaging studies in mice bearing dual flank tumors.

Main Results:

• Changes in fluorescence lifetime correlated with temperature- and time-dependent cellular internalization in vitro.
• The 1:8 conjugate demonstrated self-quenching, with fluorescence lifetime changing upon dequenching.
• In vivo imaging studies showed minimal differences in FLI between tumor types.

Conclusions:

• Fluorescence lifetime imaging (FLI) can effectively monitor the internalization of target-specific activatable antibody-fluorophore conjugates in vitro.
• Challenges exist in adapting FLI methodology for accurate in vivo imaging of these probes.