Target-cancer-cell-specific activatable fluorescence imaging probes: rational design and in vivo applications

Abstract

Conventional imaging methods, such as angiography, computed tomography (CT), magnetic resonance imaging (MRI), and radionuclide imaging, rely on contrast agents (iodine, gadolinium, and radioisotopes, for example) that are "always on." Although these indicators have proven clinically useful, their sensitivity is lacking because of inadequate target-to-background signal ratio. A unique aspect of optical imaging is that fluorescence probes can be designed to be activatable, that is, only "turned on" under certain conditions. These probes are engineered to emit signal only after binding a target tissue; this design greatly increases sensitivity and specificity in the detection of disease. Current research focuses on two basic types of activatable fluorescence probes. The first developed were conventional enzymatically activatable probes. These fluorescent molecules exist in the quenched state until activated by enzymatic cleavage, which occurs mostly outside of the cells. However, more recently, researchers have begun designing target-cell-specific activatable probes. These fluorophores exist in the quenched state until activated within targeted cells by endolysosomal processing, which results when the probe binds specific receptors on the cell surface and is subsequently internalized. In this Account, we present a review of the rational design and in vivo applications of target-cell-specific activatable probes. In engineering these probes, researchers have asserted control over a variety of factors, including photochemistry, pharmacological profile, and biological properties. Their progress has recently allowed the rational design and synthesis of target-cell-specific activatable fluorescence imaging probes, which can be conjugated to a wide variety of targeting molecules. Several different photochemical mechanisms have been utilized, each of which offers a unique capability for probe design. These include self-quenching, homo- and hetero-fluorescence resonance energy transfer (FRET), H-dimer formation, and photon-induced electron transfer (PeT). In addition, the repertoire is further expanded by the option for reversibility or irreversibility of the signal emitted through these mechanisms. Given the wide range of photochemical mechanisms and properties, target-cell-specific activatable probes have considerable flexibility and can be adapted to specific diagnostic needs. A multitude of cell surface molecules, such as overexpressed growth factor receptors, are directly related to carcinogenesis and thus provide numerous targets highly specific for cancer. This discussion of the chemical, pharmacological, and biological basis of target-cell-specific activatable imaging probes, and methods for successfully designing them, underscores the systematic, rational basis for further developing in vivo cancer imaging.

FIGURE 1

(a) A schema demonstrating targeted cancer imaging with a conventional “always on” probe (left) compared to a target-specific activatable probe (right). (b) A schema demonstrating target signal-to-background ratio of a conventional “always on” probe (left) is compared with a target-cell specific activatable probe (right).

FIGURE 2

A schema for the activation process involved with a target-cancer cell specific activatable molecular probe based on an anti growth factor receptor monoclonal antibody conjugated with an activatable fluorophore. Note that the agent is non-fluorescent in the unbound state and becomes irreversibly activated within the target cell.

FIGURE 3

A schematic explanation of the function of a target-cancer cell specific activatable probe (left) versus an enzyme activatable probe (right). The signal activation of target-cell specific activatable probes occurs intracellularly, whereas enzyme activation typically occurs in the extracellular environment permitting diffusion away from the target cell.

FIGURE 4

The schematic explanation for five available photo-chemical activation strategies; (a) self-quenching (Homo-FRET), (b) quencher-fluorophore combination, (c) auto-quenching, (d) H-type dimer formation, (e) photon induced electron transfer (PeT), and (f) a dual functional activatable fluorophore based on the combination of H-type dimer formation and PeT.

FIGURE 5

Comparison of in vivo imaging utilizing an “always on” probe (right) and a cancer cell-specific activatable probe (left). (a) A mouse model of metastatic HER2-positive lung cancer imaged 1 day after intravenous injection of “always on” (left) and activatable (right) fluorescence-labeled trastuzumab (monoclonal antibody against the HER2 receptor). The target-cell specific activatable probe (right) exclusively images targeted lung metastasis (white arrow) without visible background signal remaining unbound reagent within the blood pool. In contrast, the “always on” probe images lung cancer metastases, yet considerable background signal results from unbound agent within the blood vessels (yellow dashed arrow) and heart (H). (b) A mouse model of ovarian cancer with disseminated peritoneal metastases imaged using a fluorescence endoscopy system, 2 hours after intraperitoneal injection of “always on” (left) or activatable (right) fluorescence-labeled D-galactose lectin binding probe. The target-cell specific activatable probe (right) only shows targeted peritoneal tumors (white arrow) without background signal from unbound reagent. In contrast, the “always on” probe demonstrates higher signal from unbound reagent (yellow dashed arrow) than that from targeted tumors (white arrow).

FIGURE 6

Fluorescence imaging depicting real-time therapeutic effects in a peritoneal ovarian cancer model using a reversible pH-sensitive targeted activatable probe. The fluorescence signal derived from the activatable probe decreased with time indicating successful treatment.

FIGURE 7

A summary of the potential designs for target-cancer cell specific activatable fluorescence probes.