Delivery of molecular-specific optical contrast agents for cancer biomarker detection in live cells and tissues
van de Ven, Anne Louise
Doctor of Philosophy
Molecular-specific optical contrast agents have shown promise as potential non-invasive probes for the detection of cancer and its precursors. The topical use of optical contrast agents in vivo has been hindered, however, by the difficulty of delivering macromolecules through mucosal tissue. My goal was to develop a robust strategy for the delivery of molecular-specific optical contrast agents into live cells and tissues. Specifically, I sought to: (1) develop an efficient and reproducible strategy for intracellular delivery of optical contrast agents into live cells, (2) evaluate the feasibility of targeting human telomerase reverse transcriptase (hTERT) in live cells and fresh tissues, and (3) translate intracellular delivery strategies for the topical permeation of multi-layer mucosal tissue. This dissertation describes the development of a surfactant-based strategy to effectively and reproducibly label cancer biomarkers in live cells and tissues. Triton-X100 was evaluated for its ability to deliver targeted and untargeted optical contrast agents to different cell compartments. My findings indicate that Triton-X100, when used at the appropriate concentration, can permeabilize a variety of live cells in a reproducible and reversible manner. To assess the usefulness of Triton-X100 for the delivery molecular-specific contrast agents, antibodies specific to hTERT were delivered into live permeabilized cells. The sensitivity of this approach was validated using cell lines that differentially express hTERT and paired clinically normal and abnormal human biopsies. The feasibility of enhancing tissue permeation with Triton-X100 was assessed in freshly excised mucosal specimens. The depth and rate of tissue permeation following topical Triton-X100 treatment was evaluated as function of optical probe size. Delivery of molecular-specific optical contrast agents was tested in xenograft tumor specimens co-treated with Triton-X100. These experiments revealed that Triton-X100 can facilitate simultaneous labeling of clinically relevant intracellular and extracellular biomarkers in a controlled, uniform manner. Together, these findings provide evidence that cell- and tissue-impermeant contrast agents can be delivered into mucosal tissue in a sufficiently controlled and uniform manner to allow for cancer biomarker detection. Further studies are proposed to establish the safety of Triton-X100 for topical use in vivo .