Abstract
The conjugation of anti-cancer drugs to endogenous ligands has proven to be an effective strategy to enhance their pharmacological selectivity and delivery towards neoplasic tissues. Since cell proliferation has a strong requirement for iron, cancer cells express high levels of transferrin receptors (TfnR), making its ligand, transferrin (Tfn), of great interest as a delivery agent for therapeutics. However, a critical gap exists in the ability to non-invasively determine whether drugs conjugated to Tfn are internalized into target cells in vivo. Due to the enhanced permeability and retention (EPR) effect, it remains unknown whether these Tfn-conjugated drugs are specifically internalized into cancer cells or are localized non-specifically as a result of a generalized accumulation of macromolecules near tumors. By exploiting the dimeric nature of the TfnR that binds two molecules of Tfn in close proximity, we utilized a Förster Resonance Energy Transfer (FRET) based technique that can discriminate bound and internalized Tfn from free, soluble Tfn. In order to non-invasively visualize intracellular amounts of Tfn in tumors through live animal tissues, we developed a novel near infrared (NIR) fluorescence lifetime FRET imaging technique that uses an active wide-field time gated illumination platform. In summary, we report that the NIR fluorescence lifetime FRET technique is capable of non-invasively detecting bound and internalized forms of Tfn in cancer cells and tumors within a live small animal model, and that our results are quantitatively consistent when compared to well-established intensity-based FRET microscopy methods used in in vitro experiments.
Highlights
A major challenge in optimizing drug delivery is the ability to non-invasively monitor and quantify drug internalization into targets within live subjects. This is especially difficult in diseases such as cancer due to the enhanced permeability and retention (EPR) effect that results in the accumulation of macromolecules in the region of the tumor caused by increased tumor vascular permeability and decreased lymphatic drainage [1,2]
We have identified and characterized a fluorophore pair AlexaFluor700 (AF700) and AlexaFluor 750 (AF750) in the near infrared (NIR) range with sufficient brightness, stability, lifetime contrast, while being capable of achieving a high efficiency of Forster Resonance Energy Transfer (FRET) due to a strong R0 Forster distance [32]
As an alternative to visible and intensity-based FRET microscopy, we developed a NIR fluorescence lifetime FRET macroscope system that detects FRET based on the reduction of donor fluorophore lifetime [33]
Summary
A major challenge in optimizing drug delivery is the ability to non-invasively monitor and quantify drug internalization into targets within live subjects. This is the first report to use NIR fluorescence lifetime FRET to non-invasively detect Tfn internalization in human cancer cells through living animal tissues.
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