Near-Infrared Fluorescent Peptide Probes for Imaging Cancer

Cancer imaging is useful for multiple clinical and research purposes such as diagnostics, surgery guidance, monitoring disease progression, and metastasis detection. In particular, fluorescence imaging has the advantage of being relatively non-toxic and inexpensive compared to other imaging modalities, and thus, there is a need to create new classes of cancer-targeted fluorescent probes. This thesis describes cancer imaging studies using novel molecular probes based on two near-infrared dyes called squaraine dyes and heptamethine cyanine dyes.

The first half of Chapter 1 provides a broad introduction to the different strategies that are used to fabricate molecular probes. Over the last decade, covalent click chemistry has emerged as a powerful synthetic method for connecting two molecules together to make a larger conjugate for molecular imaging. Also discussed is the new concept of non-covalent click chemistry as an effective way to conjugate molecules rapidly and under mild conditions. The second half of Chapter 1 provides a broad introduction to the concept of targeted cancer imaging using targeted molecular probes. The focus is on aminopeptidase N as a molecular protein target that is overexpressed on the surface of cancer cells and tumor vasculature. Many different imaging modalities have been investigated in recent years and the work is presented as a classic example of systematic progress in modern molecular imaging.

The second chapter describes an imaging study using a new squaraine fluorescent probe with a covalently attached cyclized peptide sequence Arg-Gly-Asp-D-Phe-Lys (cRGDfK). The cRGDfK peptide targets integrin receptors overexpressed on the surface of cancer cells and tumor vasculature. The probe was utilized to fluorescently image a clinically relevant mouse tumor model of ovarian cancer. In the second half of the chapter, the cRGDfK peptide was incorporated onto a tetralactam macrocycle which subsequently threaded a squaraine dye. The pre-assembled probe was used to successfully target implanted tumors comprised of non-small cell lung cancer cells. The third chapter summarizes a study using a new non-covalent cRGDfK probe with a different tetralactam macrocycle and squaraine dye. This probe permitted successful targeting of a cancerous tumor in a mouse model and enabled a mock surgery that removed the tumor. However, the tumor-to-background ratio varied significantly between mice due to tumor-to-tumor variability of the vasculature structure and interstitial pressure. Thus, a new paired agent imaging technique was developed where squaraine dyes with two different emission wavelengths were encapsulated within either a cRGDfK macrocycle or an untargeted macrocycle. The non-covalent probes were simultaneously injected into tumor-bearing mice and imaging showed enhanced tumor accumulation of the targeted probe with less tumor-to-tumor variability.

The fourth chapter describes a new probe molecule whose structure is based on a figure-eight scaffold. The scaffold permits easy synthetic incorporation of peptide loops as rigid recognition motifs for selective, high affinity biological targeting. The squaraine figure-eight probes displayed excellent imaging capabilities for in-vitro cancer imaging, histological bone staining, and in-vivo bone imaging. The last chapter focuses on a new class of non-aggregating heptamethine cyanine dyes which have short ethylene glycol chains that serve as protecting “arms” that shields the hydrophobic core. These dyes were used for antibody bioconjugation and in-vivo cancer imaging. They displayed ideal in-vivo imaging characteristics such as low serum binding, low cell toxicity, good water solubility, and safe and complete in-vivo clearance.