We describe multimodal nonlinear microscopy using a compact, turnkey femtosecond fiber laser at 1.5 μm. The system
allows for multiplexed detection of near infrared and visible contrast agents through two-and three-photon excitation
fluorescence microscopy as well as structural imaging viaauto-confocal microscopy (ACM). This platform expands the
available emission spectrum for multiphoton microscopy, enables simultaneous structural and functional imaging, and
offers advantages in penetration depth, contrast, and simplicity as compared to conventional MPM near 800 nm
excitation.
Local molecular and physiological processes can be imaged in vivo through perturbations in the fluorescence lifetime (FLT) of optical imaging agents. In addition to providing functional information, FLT methods can quantify specific molecular events and multiplex diagnostic and prognostic information. We have developed a fluorescence lifetime diffuse optical tomography (DOT) system for in vivo preclinical imaging. Data is captured using a time-resolved intensified charge coupled device (ICCD) system to measure fluorescence excitation and emission in the time domain. Data is then converted to the frequency domain, and we simultaneously reconstruct images of yield and lifetime using an extension to the normalized Born approach. By using differential phase measurements, we demonstrate DOT imaging of short lifetimes (from 350 ps) with high precision (±5 ps). Furthermore, this system retains the efficiency, speed, and flexibility of transmission geometry DOT. We demonstrate feasibility of FLT-DOT through a progressive series of experiments. Lifetime range and repeatability are first measured in phantoms. Imaging of subcutaneous implants then verifies the FLT-DOT approach in vivo in the presence of inhomogeneous optical properties. Use in a common research scenario is ultimately demonstrated by imaging accumulation of a targeted near-infrared (NIR) fluorescent-labeled peptide probe (cypate-RGD) in a mouse with a subcutaneous tumor.
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.
Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.
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.
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.
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