Colitis-associated cancer (CAC) arises from premalignant flat lesions of the colon, which are difficult to detect with current endoscopic screening approaches. We have developed a complementary fluorescence and polarization reporting strategy that combines the unique biochemical and physical properties of dysplasia and cancer for real-time detection of these lesions. Using azoxymethane-dextran sodium sulfate (AOM-DSS) treated mice, which recapitulates human CAC and dysplasia, we show that an octapeptide labeled with a near-infrared (NIR) fluorescent dye selectively identified all precancerous and cancerous lesions. A new thermoresponsive sol-gel formulation allowed topical application of the molecular probe during endoscopy. This method yielded high contrast-to-noise ratios (CNR) between adenomatous tumors (20.6±1.65) and flat lesions (12.1±1.03) and surrounding uninvolved colon tissue versus CNR of inflamed tissues (1.62±0.41). Incorporation of nanowire-filtered polarization imaging into NIR fluorescence endoscopy shows a high depolarization contrast in both adenomatous tumors and flat lesions in CAC, reflecting compromised structural integrity of these tissues. Together, the real-time polarization imaging provides real-time validation of suspicious colon tissue highlighted by molecular fluorescence endoscopy.
The clinical diagnosis of most cancers is based on evaluation of histology microscopic slide to view the size and shape of
cellular nuclei, and morphological structure of tissue. To achieve this goal in vivo and in deep tissue, near infrared (NIR)
dyes-bovine serum albumin (BSA) and immunoglobulin G (IgG) conjugates were synthesized. The spectral study show
that the absorption and fluorescence of the dye-conjugates are in the “tissue optical window” between 650 nm and 1100
nm. The internalization and pinocytosis of the synthesized compound were investigated in cell level using fluorescence
microscopy to obtain the optimal concentration and staining time scale.
A series of novel near-infrared fluorescent compounds containing both desferrioxamine (DFO) and multi-RGD
peptides, i.e. DFO-Cypate-(RGD)n-NH2 (1), were designed and synthesized based on a dicarboxylic acid-containing
near-infrared fluorescent carbocyanine (Cypate) scaffold. The trimeric 1 (n=3) showed the strongest cellular
internalization into A549 cells in vitro among the four analogs of 1 (n=1, 2, 3, 4), suggesting that such a linear array of
three RGD peptide motifs might be optimal for synergistic effects on cellular internalization. The four analogs showed
higher internalization than an integrin αvβ3-targeting cyclic RGD peptide analog DFO-cypate-[RGDfK(~)] (2) after 1h
of incubation, indicating that the linear arrays of multi-RGD peptides might be different from the cyclic RGD peptide
analog in the internalization kinetics and mechanism of receptor targeting. Confocal microscopy showed that 1 (n=4)
could localize at least in part to the mitochondria. Noteworthy, the two compounds 1 (n=2, 3) resulted in a 1.5 to 2 fold
increase in fluorescence of the calcium indicator fluo4 after 30 min of incubation. These results suggest the possible
effects of these compounds on the cellular function by internalization. Such a type of near-infrared fluorescent cypate
analogs containing both DFO and multi-RGD peptides could provide a platform for discovering and developing novel
multifunctional optical contrast agents for integrin receptor targeting as well as related tumor imaging and therapy.
In order to explore novel NIR fluorescent probes for optical imaging in biomedicines, one desferrioxamine
(DFO)-bearing NIR fluorescent probe was designed and synthesized based on a dicarboxylic acid-containing
carbocyanine (Cypate). Similar to the free DFO, the resulting conjugate Cypate-DFO showed high binding affinity with
Fe(III) and Ga(III) as identified by ES-MS. Nevertheless, the iron binding was found to quench its fluorescent emission
significantly, suggesting that the siderophore moiety might perturb the spectroscopic properties of the attached
carbocyanine fluorophore through metal binding. As observed by fluorescence microscopy, Cypate-DFO showed
significant cellular internalization in A549 cells in vitro. Further studies on novel Cypate-DFO derivatives of this type
may reveal some exciting properties and biological activities.
Expression of integrin αvβ3 is upregulated in a number of cancers including colon, pancreas, lung and breast. Previous studies demonstrated that near infrared (NIR) fluorescent probes designed to target αvβ3 accumulated both in vitro and in vivo in αvβ3-positive tumor cells. To evaluate the selectivity of some NIR-labeled RGD peptides for αvβ3, the molecular probes were incubated in different cells, including the αvβ3-positive U87 and A549 cells, and αvβ3-negative HT29 cells. Whereas the RGD compounds tested internalized in the A549 cells, their uptake by the HT29 cell line, which is positive for αvβ5 and αvβ6, was low. The uptake of these probes in U87 depended on the structural features of the compounds. Further studies with functional blocking antibodies showed that the internalization in the αvβ3-positive cells may be mediated by different integrin receptor subtypes. The preliminary results suggest that the internalization of linear RGD peptides is mediated by the αvβ3 heterodimer but rearrangement of the peptide sequence could alter the selectivity of the molecular probes for different integrin subunits in the dimeric α and β proteins. Thus, a careful choice of RGD peptides can be used to monitor the functional status of different integrins in cells and tissues.
KEYWORDS: Near infrared, Receptors, Magnesium, Luminescence, In vivo imaging, Tumors, Optical imaging, Analog electronics, Chemistry, Biomedical optics
Interest in novel RGD peptides has been increasingly growing as the interactions between RGD peptides and integrins are the basis for a variety of cellular functions and medical applications such as modulation of cell adhesion, invasion, tumor angiogenesis, and metastasis. In particular, we have been interested in novel NIR fluorescent RGD peptides as potential optical contrast agents for in vivo tumor optical imaging. Therefore, two cyclic RGD penta-peptides conjugated with a NIR fluorescent carbocyanine (Cypate), i.e. lactam-based cyclo[RGDfK(Cypate)] (1) and disulfide-containing Cypate-cyclo(CRGDC)-NH2 (2), were designed and synthesized. The competitive binding assay between the purified αvβ3 integrin and the peptide ligands using 125I-echistatin as a tracer showed that 1 had a higher receptor binding affinity (IC50~10-7 M) than 2 (IC50~10-6 M). Furthermore, the internalization of 1 in A549 cells in vitro was less than 2, as revealed by fluorescence microscopy. These results suggest that both the lactam- and disulfide-based cyclic RGD penta-peptides should be further studied structurally and functionally to elucidate the advantages of each class of compounds.
KEYWORDS: Tumors, Luminescence, Tissues, Fluorescence lifetime imaging, Near infrared, Liver, In vivo imaging, Kidney, Optical imaging, Imaging systems
Fluorescence lifetime imaging can provide valuable diagnostic information relating to the functional status of diseases. In this study, a near-infrared (NIR) dye-labeled hexapeptide (abbreviated Cyp-GRD) was synthesized. In vitro, Cyp-GRD internalized in nonsmall cell lung cancer cells (A549) without observable cytotoxic or proliferative effects to the cells at a concentration up to 1×10–4 M. Time-domain fluorescence intensity and lifetime imaging of Cyp-GRD injected into A549 tumor-bearing mice revealed that the probe preferentially accumulated in the tumor and the major excretion organs. The fluorescence lifetime of the conjugate at the tumor site was mapped, showing the spatial distribution of the lifetime related to its environment. Additionally, fluorescence intensity image reconstruction obtained by integrating the time-resolved intensities enabled the contrast ratios of tumor-to-kidney or liver in slices at different depths to be displayed. The mean lifetime was 1.03 ns for the tumor and 0.80 ns for the liver when averaging those pixels exhibiting adequate signal-to-noise ratio, showing the tumor had a higher lifetime average and reflecting the altered physiopathology of the tumor. This study clearly demonstrated the feasibility of whole-body NIR fluorescence lifetime imaging for tumor localization and its spatial functional status in living small animals.
Expression of integrin αvβ3 is upregulated in a number of cancers including colon, pancreas, lung and breast. Additionally, αvβ3 integrin expression has been linked to tumor metastasis and targeting this cell surface protein could provide a viable approach to image and evaluate the metastatic potential of tumors. Accordingly, we evaluated the selective retention of some near infrared (NIR) fluorescent probes in nude mice bearing A549 lung cancer xenograft that express αvβ3 integrin. Our preliminary results indicate that a novel NIR probe designed to target this integrin selectively accumulated in A549 tumor while other non-integrin specific probes were not retained in the tumor. Blocking studies show that tumor uptake of the probe is mediated by αvβ3 integrin receptor.
A new carbocyanine optical molecular probe with enhanced water solubility and constrained structural conformations was designed and synthesized. The near infrared (NIR) fluorescent probe contains a nonionic D-galactopyranose, which could improve water solubility of the probe and enhance uptake in tumors mediated by glucose transporter. The possibility of multiple attachment points provides the potential to conjugate diverse bioactive molecules to the probe. We developed an efficient synthetic method that is optimized for large-scale synthesis. Preliminary in vivo biodistribution studies show that the probe is rapidly cleared from blood and localize in the liver as early as 5 minutes post-injection of the probe in nude mice. Additional studies to evaluate the tumor uptake of the probe and its bioactive peptide conjugates are in progress.
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