Development of EGFR-targeted contrast agents for in vivo applications
Nida, Dawn Lynn
Doctor of Philosophy
Optical techniques targeting biomarkers of cancer have the potential to aid in the early detection of this disease. Molecular and biochemical changes in neoplastic tissue could be interrogated, potentially providing an objective assessment and identification of suspect tissue. A method of interrogating such changes involves the development of optical based probes that provide a molecular-specific source of signal. This dissertation aims to develop an effective optical strategy to target the epidermal growth factor for in vivo detection. The optical probe design consists of an optically active component (quantum dot, fluorescent dye) and a molecular specific probe (antibody, peptide). The optically active components investigated were cadmium telluride quantum dots (QDs) and Alexa Fluor 647 dye. Epidermal growth factor receptor (EGFR) was chosen as a molecular target because it is over-expressed in a large number of epithelial cancers. Both peptides and antibody to EGFr were investigated as molecular specific probes. The work presented in this dissertation provides a systematic approach for developing optically active probes targeting EGFR. The ability to synthesize, passivate and functionalize red-emitting QDs was investigated. It was demonstrated that in order for QDs to be properly passivated for biological imaging applications, a crosslinked amphiphilic-based polymer should be used. Such polymers afforded the best surface protection of all the coating strategies tested. Unfortunately, such systems are generally very large (>35nm) and therefore might be hindered in in vivo delivery. Due to the large size of QD-based optical probes, further research in this dissertation utilized the commercially available dye, Alexa Fluor 647. Alexa 647 was tethered individually to three different proteins: anti-EGFR antibody; mouse epidermal growth factor peptide (mEGF); and human epidermal growth factor peptide (hEGF). The specificity of each Alexa 647-protein conjugate was verified in vitro. Each protein-dye conjugate was further investigated to determine the specificity and efficiency of intravenous delivery to EGFR positive and EGFR negative tumors. These investigations revealed the importance of both the mean fluorescent signal (normalized for dye:protein ratio) as well as distinctive, recognizable patterns of contrast agents for in vivo applications. Such criteria yield a contrast agent with the best case scenario for being detected and recognized. The use of a systematic approach to design and test contrast agents for in vivo applications was demonstrated through this work. This work stressed a comprehensive investigation including in vitro, ex vivo and in vivo testing. Additionally, it was shown that care must be taken in designing in vitro experiments to interpret in vivo results as the two do not necessarily correlate. This dissertation lays the groundwork for the development of contrast agents targeting specific molecular markers of cancer.