The thrust of our project stems from analysis of the current methods for the delineation of cancerous tissues using fluorescence-based in vivo imaging agents;current agents are mostly unselective for diseased tissues and cannot effectively differentiate cancerous from normal tissues. These agents also cannot detect small cancers, typically those less than 5 mm, thereby hindering early cancer detection. In order to improve upon confocal laser endomicroscopy, a newly developed method for in vivo imaging of colorectal cancers (CRC), we propose new porphyrin-based in vivo imaging agents with structural features to further increase selectivity for CRC cells, which over-express epidermal growth factor receptor (EGFR). This research proposal, used in conjunction with confocal laser endomicroscopy for CRC detection, will allow the achievement of the following specific aims: 1) Synthesize a series of porphyrin-based conjugates by selecting ligands (peptidic and non-peptidic) with high affinity for an over-expressed receptor (EGFR) at the surface of CRC cells, thereby improving their cancer cell-targeting ability. 2) To biologically evaluate the series of porphyrin- based conjugates in cell culture and, if promising, evaluate the molecules in vivo. In cell culture, conjugates will be analyzed for their phototoxicity, cellular uptake, and subcellular localization. In vivo, conjugates will be evaluated based on their toxicity, retention times, and fluorescence emission. Our proposed synthetic molecules will reduce background fluorescence from biological tissues, such as blood and plasma, as the use of many porphyrin-based molecules emit at wavelengths greater than 630 nm. This is a great comparison to the in vivo imaging agent, fluorescein (Ex. 494 nm, Em. 521 nm), which fluoresces in the visible region of the spectrum. Our molecules will also improve water-solubility, decrease toxicity, and decrease aggregation of the porphyrin conjugates, also potentially improving their biological efficacy compared with current fluorophores with the conjugation of a polyethylene glycol linker (PEG) to the porphyrin. More importantly, the cancer cell-targeting ability of our porphyrin-based molecules will be improved by selecting ligands with high affinity for an over-expressed receptor (EGFR) at the surface of CRC cells. This proposal discusses the design and synthesis of new porphyrin peptidic and non-peptidic conjugates, their characterization, and biological investigation based on preliminary investigations. In alignment with the mission of the NIH and its other agencies, this inter-disciplinary project fosters innovative research in the prevention of CRC.
A series of epidermal growth factor receptor (EGFR) porphyrin-based conjugates are being developed as a tool in the assessment of colorectal cancer cells (CRC). Despite the CRC's presence as the third leading cause of cancer deaths in the United States,1 we must utilize the biocompatibility of the highly fluorescent, stable, aromatic porphyrin, which has been extensively studied and is currently being used in medicinal applications, such as photodynamic therapy (PDT).25-35 This research, then, is advantageous in that fluorescent porphyrin-based conjugates be used to selectively and efficiently label CRC tumor foci, which has been illustrated by previous research in our group.
