Wide-field macroscopic fluorescence lifetime imaging for pre-clinical assessment of targeted drug delivery

Abstract

This study optimized our wide-field MFLI FRET imaging for preclinical applications by selecting the best near-infrared (NIR) FRET pair and implementing reflectance imaging geometry. Comparison of five NIR FRET pairs using a cell-based assay revealed that Alexa Fluor 700 and Alexa Fluor 750 was the best NIR pair due to its brightness and a good relationship between FRET level and acceptor to donor ratios. Implementation of reflectance illumination geometry allowed whole-body imaging of small animals. The validation was performed with the previously implemented transmission illumination geometry. There was no difference between lifetime-based FRET quantification in vitro and in vivo.

In summary, wide-field MFLI has been optimized for whole-body preclinical studies and validated with other traditional methods. The results confirmed that the wide-field MFLI provide unique features for preclinical assessment of targeted drug delivery.

Last, we investigated dynamic in vivo FRET applications for a longitudinal study of transferrin ligand-receptor engagement using intensity and lifetime measurements. This study confirmed that there was an agreement between both measurements for the quantification of standard DNA FRET samples. Dynamic in vivo FRET showed that transferrin ligand-receptor engagement could be monitored dynamically using both intensity and lifetime FRET measurements. However, intensity FRET measurements required calibration samples, while lifetime FRET measurement did not. The simple experiment setup allowed lifetime FRET to be used for in vivo competitive receptor binding assay, which was demonstrated by using high concentration unlabeled transferrin as a competitor. The difference in the constant kinetic rate between mice with and without the competitor was observed and quantified.

This study employed our wide-field MFLI platform for preclinical oncology applications. In vivo transferrin ligand-receptor engagement in tumor xenografts was quantified using MFLI FRET. There was a difference of FRET level between tumors and the urinary bladders, confirming that there was ligand-receptor engagement in the tumors but not in the urinary bladder, which was the probe excretion organ. MFLI FRET method was also compared with the standard histopathological evaluation. There was no correlation of FRET level with tumor sizes or the receptor expression levels. However, a positive correlation between FRET level and Tf uptake of tumors existed. Additionally, the wide-field MFLI was applied for glucose metabolic activity assessment in tumor xenografts using NIR 2-DG. It was demonstrated that both transferrin ligand-receptor engagement and glucose metabolic activity could be evaluated using the wide-field MFLI concurrently in the same animal.

Preclinical imaging can improve the efficiency of drug development process by providing drug efficacy information in pathophysiological context. Most preclinical imaging modalities only provide intensity readings, which are not sufficient to uncover drug action in vivo. Wide-field macroscopic fluorescence lifetime imaging (MFLI) can overcome the limitation by providing additional information, lifetime. Of particular interest, fluorescence lifetime can be exploited to assess intracellular delivery by using Förster Resonance Energy Transfer (FRET)-based sensing approach.