The overall goal of this project is to develop a non-invasive, high resolution imaging strategy to assess both biodistribution and cellular uptake of polymer carriers in vivo. Polymer-drug conjugates have great potential as anticancer agents, due to improved pharmacokinetics as a result of enhanced permeability and retention (EPR) effect. However, their efficacy relies not only on their increased accumulation in the tumor tissue, but also their ability to subsequently deliver their therapeutic payload to intracellular targets. As such, it is hypothesized that the ability to optimize endocytic efficiency - the fractin of conjugates in the tissue that is actually internalized by cells - should lead to better and more clinically relevant polymer-drug conjugates. However, there is no straightforward, generalizable means to non-invasively assess cellular uptake of polymer-drug conjugates in preclinical models. In order to address this shortcoming and test this hypothesis, a novel imaging strategy is proposed that uses an activatable fluorophore that is triggered upon endocytosis of the polymer-drug conjugate. The proposed design uses fluorescence resonance energy transfer (FRET) and a lysosomally-degradable peptide in order to create an activatable system. Specifically, a donor fluorophore and quencher are conjugated to the polymer-drug conjugate, but are separated by peptide substrate for lysosomal enzymes. Prior to endocytosis, the dyes are in close proximity, and exciting the donor will not induce fluorescence. However, upon encountering the lysosomal enzyme, the peptide will be cleaved, breaking the link between the dyes and freeing them to diffuse apart. The donor fluorophore will then emit a bright fluorescence upon excitation. The ability of this system to report endocytosis in vitro and in vivo will be evaluated. To conduct the in vivo imaging, fluorescence molecular tomography (FMT) will be used to obtain non-invasive whole-body 3- dimensional imaging in a small animal model, quantitatively imaging probes at picomolar levels at depths greater than one centimeter. In parallel, positron emission tomography (PET) imaging will provide information on polymer biodistribution. Then, chemotherapeutics and targeting moieties will be incorporated into the polymer design to produce a new polymer theranostic (therapeutic + diagnostic) system. Ovarian carcinoma will serve as a model disease target to validate the utility of imaging results for preclinical evaluation of polymer-drug conjugates. The ability of the proposed imaging strategy to inform selection of optimized therapeutics and enable prediction of their efficacy will be evaluated.
This proposal describes a novel multimodal imaging strategy to dynamically assess biodistribution and cellular uptake of polymer-drug conjugates in vivo. An activatable fluorescent probe that is triggered upon endocytosis will be incorporated into a polymer-drug conjugate design. Evaluation of the fluorescence data in vitro and in vivo will then be used to optimize the drug delivery system against ovarian carcinoma as a model disease.