Optical detection of tumors with a receptor-targeted fluorescent contrast agent has been demonstrated. This result represents a new direction in cancer diagnosis and patient management.

We have designed, synthesized, and evaluated the efficacy of novel dye-peptide conjugates that are receptor specific. Contrary to the traditional approach of conjugating dyes to large proteins and antibodies, we used small peptide-dye conjugates that target over-expressed receptors on tumors. Despite the fact that the peptide and the dye probe have similar molecular mass, our results demonstrate that the affinity of the peptide for its receptor and the dye fluorescence properties are both retained. The use of small peptides has several advantages over large biomolecules, including ease of synthesis of a variety of compounds for potential combinatorial screening of new targets, reproducibility of high purity compounds, diffusiveness to solid tumors, and the ability to incorporate a variety of functional groups that modify the pharmacokinetics of the peptide-dye conjugates. The efficacy of these new fluorescent optical contrast agents was evaluated in vivo in well-characterized rat tumor lines expressing somatostatin (sst(2)) and bombesin receptors. A simple continuous wave optical imaging system was employed. The resulting optical images clearly show that successful specific tumor targeting was achieved. Thus, we have demonstrated that small peptide-dye conjugates are effective as contrast agents for optical imaging of tumors.

Site-specific delivery of drugs and contrast agents to tumors protects normal tissues from the cytotoxic effects of drugs and enhances the contrast between normal and pathologic tissues. One approach to achieve selectivity is to target overexpressed receptors on the membranes of tumor cells and to visualize the tumors by a noninvasive optical imaging method. Accordingly, we conjugated fluorescein and carbocyanine dyes to somatostatin and bombesin receptor-avid peptides and examined their receptor binding affinities. We also prepared potential dual imaging probes consisting of a bioactive peptide for tumor targeting, a biocompatible dye for optical imaging, and a radioactive or paramagnetic metal chelator for scintigraphic or magnetic resonance imaging of tumors. Using these approaches, the resulting carbocyanine derivatives of somatostatin and bombesin analogues retained high binding for their respective receptors. Further evaluation of representative molecules in rats bearing somatostatin- and bombesin-positive tumors showed selective uptake of the agents by the tumor cells. Unlike carbocyanine derivatives, the receptor binding of fluorescein-somatostatin peptide conjugates was highly sensitive to the type of linker and the site of fluorescein attachment on the nonreceptor binding region of the peptide. In general, the presence of flexible linkers disrupted binding affinity, possibly due to the interaction of the linker's thiourea group with the peptide's cyclic disulfide bond. While the receptor binding affinity of the dual probes was not dependent on the type of chelating group examined, it was affected by the relative positions of fluorescein and chelator on the lysine linker. For somatostatin compounds, best results were obtained when the chelator was on the alpha-amino lysine linker and fluorescein was on the epsilon-amino group. In contrast, conjugation of the chelator to epsilon- and fluorescein to the alpha-amino lysine linker of bombesin peptides resulted in high receptor binding. These findings indicate that despite their small size, conjugation of dyes to truncated somatostatin and bombesin peptide analogues results in promising diagnostic agents that retain high receptor binding activity in vitro. The results further show that these contrast agents can selectively and specifically localize in receptor-positive tumors in rat models.

mouse ͉ optical imaging ͉ RGD peptides ͉ tumor ͉ near-infrared A ngiogenesis, the formation of new blood vessels, is the cardinal feature of virtually all malignant tumors (1). Because of this commonality, probing tumor-induced angiogenesis and associated proteins is a viable approach to detect and treat a wide range of cancers. Angiogenesis is stimulated by integrins, a large family of transmembrane proteins that mediate dynamic linkages between extracellular adhesion molecules and the intracellular actin skeleton. Integrins are composed of two different subunits, ␣ and ␤, which are noncovalently bound into ␣␤ complexes (2-4). Particularly, the expression of ␣ v ␤ 3 integrin (ABI) in tumor cells undergoing angiogenesis and on the epithelium of tumor-induced neovasculature alters the interaction of cells with the extracellular matrix, thereby increasing tumorigenicity and invasiveness of cancers (5-9).Numerous studies have shown that ABI and more than seven other heterodimeric integrins recognize proteins and low molecular weight ligands containing RGD (arginine-glycineaspartic acid) motifs in proteins and small peptides (10). Based on structural and bioactivity considerations, cyclic RGD peptide ligands are preferentially used as delivery vehicles for molecular probes for imaging (8,(11)(12)(13) and treating (14-17) ABI-positive tumors and proliferating blood vessels. Until recently, most of the in vivo imaging studies were performed with radiopharmaceuticals because of the high sensitivity and clinical utility of nuclear imaging methods. Particularly, the use of small monoatomic radioisotopes does not generally interfere with the biodistribution and bioactivity of ligands. Despite these advantages, nuclear imaging is currently only performed in specialized centers because of regulatory, production, and handling issues associated with radiopharmaceuticals. Optical imaging is an alternative but complementary method to interrogate molecular processes in vivo and in vitro.Optical imaging for biomedical applications typically relies on activating chromophore systems with low energy radiation between 400 -and 1,500-nm wavelengths and monitoring the propagation of light in deep tissues with a charge-coupled device camera or other point source detectors (18). Molecular optical imaging of diseases with molecular probes is attractive because of the flexibility of altering the detectable spectral properties of the probes, especially in the fluorescence detection mode. The probes can be designed to target cellular and molecular processes at functional physiological concentrations. For deep-tissue imaging, molecular probes that are photoactive in the near-infrared (NIR) instead of visible wavelengths are preferred to minimize background tissue autof luorescence and light attenuation caused by absorption by intrinsic chromophores (19). In contrast to radioisotopes, the NIR antennas are usually large heteroatomic molecules that could impact the biodistribution and activity of conjugated bioactive ligands. However, previous s...

A new type of degradable, nanoscopic polymer assembly containing ultra-high levels of drug loading via covalent attachment within amphiphilic core-shell nanoparticle morphology has been generated as a potentially effective and safe anti-cancer agent. Poly(ethylene oxide)-block-polyphosphoester-based paclitaxel drug conjugates (PEO-b-PPE-g-PTX) were synthesized by rapid, scalable and versatile approach that involves only two steps: organocatalyst-promoted ring-opening-polymerization followed by click reaction-based conjugation of a PTX prodrug. Variations in the polymer-to-PTX stoichiometries allowed for optimization of the conjugation efficiency, the PTX drug loading and the resulting water solubilities of the entire polymer and the PTX content. The PEO-b-PPE-g-PTX formed well-defined micelles in aqueous solution, with a PTX loading capacity as high as 65 wt%, and a maximum PTX concentration of 6.2 mg/mL in water, which is 25000-fold higher than the aqueous solubility of free PTX. The positive cell-killing activity of PEO-b-PPE-g-PTX against several cancer cell lines is demonstrated, and the presence of pendant reactive functionality provides a powerful platform for future work to involve conjugation of multiple drugs and imaging agents to achieve chemotherapy and bioimaging.