Selective imaging of solid tumours via the calcium-dependent high-affinity binding of a cyclic octapeptide to phosphorylated Annexin A2.

The heterogeneity and continuous genetic adaptation of tumours complicate their detection and treatment via the targeting of genetic mutations. However, hallmarks of cancer such as aberrant protein phosphorylation and calcium-mediated cell signalling provide broadly conserved molecular targets. Here, we show that, for a range of solid tumours, a cyclic octapeptide labelled with a near-infrared dye selectively binds to phosphorylated Annexin A2 (pANXA2), with high affinity at high levels of calcium. Because of cancer-cell-induced pANXA2 expression in tumour-associated stromal cells, the octapeptide preferentially binds to the invasive edges of tumours and then traffics within macrophages to the tumour's necrotic core. As proof-of-concept applications, we used the octapeptide to detect tumour xenografts and metastatic lesions, and to perform fluorescence-guided surgical tumour resection, in mice. Our findings suggest that high levels of pANXA2 in association with elevated calcium are present in the microenvironment of most solid cancers. The octapeptide might be broadly useful for selective tumour imaging and for delivering drugs to the edges and to the core of solid tumours.

Acidic extracellular pH of tumors induces octamer-binding transcription factor 4 expression in murine fibroblasts in vitro and in vivo.

Octamer-binding transcription factor 4 (OCT-4) is an important marker of cellular de-differentiation that can be induced by environmental stressors, such as acidity. Here we demonstrate that chronic acidic stress in solid tumors induced OCT-4 expression in fibroblasts and other stromal cells in four tumor models. The results have implications for how tumors utilize pH modulation to recruit associated stromal cells, induce partial reprogramming of tumor-associated stromal cells, and respond to therapy.

Visualization of pulmonary clearance mechanisms via noninvasive optical imaging validated by near-infrared flow cytometry.

In vivo optical imaging with near-infrared (NIR) probes is an established method of diagnostics in preclinical and clinical studies. However, the specificities of these probes are difficult to validate ex vivo due to the lack of NIR flow cytometry. To address this limitation, we modified a flow cytometer to include an additional NIR channel using a 752 nm laser line. The flow cytometry system was tested using NIR microspheres and cell lines labeled with a combination of visible range and NIR fluorescent dyes. The approach was verified in vivo in mice evaluated for immune response in lungs after intratracheal delivery of the NIR contrast agent. Flow cytometry of cells obtained from the lung bronchoalveolar lavage demonstrated that the NIR dye was taken up by pulmonary macrophages as early as 4-h post-injection. This combination of optical imaging with NIR flow cytometry extends the capability of imaging and enables complementation of in vivo imaging with cell-specific studies.

Trimodal color-fluorescence-polarization endoscopy aided by a tumor selective molecular probe accurately detects flat lesions in colitis-associated cancer.

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±0.42) 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.

Imaging mRNA expression levels in living cells with PNA·DNA binary FRET probes delivered by cationic shell-crosslinked nanoparticles.

Optical imaging of gene expression through the use of fluorescent antisense probes targeted to the mRNA has been an area of great interest. The main obstacles to developing highly sensitive antisense fluorescent imaging agents have been the inefficient intracellular delivery of the probes and high background signal from unbound probes. Binary antisense probes have shown great promise as mRNA imaging agents because a signal can only occur if both probes are bound simultaneously to the mRNA target site. Selecting an accessible binding site is made difficult by RNA folding and protein binding in vivo and the need to bind two probes. Even more problematic, has been a lack of methods for efficient cytoplasmic delivery of the probes that would be suitable for eventual applications in vivo in animals. Herein we report the imaging of iNOS mRNA expression in live mouse macrophage cells with PNA·DNA binary FRET probes delivered by a cationic shell crosslinked knedel-like nanoparticle (cSCK). We first demonstrate that FRET can be observed on in vitro transcribed mRNA with both the PNA probes and the PNA·DNA hybrid probes. We then demonstrate that the FRET signal can be observed in live cells when the hybrid probes are transfected with the cSCK, and that the strength of the FRET signal is sequence specific and depends on the mRNA expression level.

Near-infrared fluorescent divalent RGD ligand for integrin αvß3-targeted optical imaging.

A new near-infrared fluorescent compound containing two cyclic RGD motifs, cypate-[c(RGDfK)](2) (1), was synthesized based on a carbocyanine fluorophore bearing two carboxylic acid groups (cypate) for integrin α(v)ß(3)-targeting. Compared with its monovalent counterpart cypate-c(RGDfK) (2), 1 exhibited remarkable improvements in integrin α(v)ß(3) binding affinity and tumor uptake in nude mice of A549. The results suggest that cypate-linked divalent ligands can serve as an important molecular platform for exploring receptor-targeted optical imaging and treatment of various diseases.

Near-infrared molecular probes for in vivo imaging.

Cellular and tissue imaging in the near-infrared (NIR) wavelengths between 700 and 900 nm is advantageous for in vivo imaging because of the low absorption of biological molecules in this region. This unit presents protocols for small animal imaging using planar and fluorescence lifetime imaging techniques. Included is an overview of NIR fluorescence imaging of cells and small animals using NIR organic fluorophores, nanoparticles, and multimodal imaging probes. The development, advantages, and application of NIR fluorescent probes that have been used for in vivo imaging are also summarized. The use of NIR agents in conjunction with visible dyes and considerations in selecting imaging agents are discussed. We conclude with practical considerations for the use of these dyes in cell and small animal imaging applications.

Near-infrared pH-activatable fluorescent probes for imaging primary and metastatic breast tumors.

Highly tumor selective near-infrared (NIR) pH-activatable probe was developed by conjugating pH-sensitive cyanine dye to a cyclic arginine-glycine-aspartic acid (cRGD) peptide targeting α(v)ß(3) integrin (ABIR), a protein that is highly overexpressed in endothelial cells during tumor angiogenesis. The NIR pH-sensitive dye used to construct the probe exhibits high spectral sensitivity with pH changes. It has negligible fluorescence above pH 6 but becomes highly fluorescent below pH 5, with a pK(a) of 4.7. This probe is ideal for imaging acidic cell organelles such as tumor lysosomes or late endosomes. Cell microscopy data demonstrate that binding of the cRGD probe to ABIR facilitated the endocytosis-mediated lysosomal accumulation and subsequent fluorescence enhancement of the NIR pH-activatable dye in tumor cells (MDA-MB-435 and 4T1/luc). A similar fluorescence enhancement mechanism was observed in vivo, where the tumors were evident within 4 h post injection. Moreover, lung metastases were also visualized in an orthotopic tumor mouse model using this probe, which was further confirmed by histologic analysis. These results demonstrate the potential of using the new integrin-targeted pH-sensitive probe for the detection of primary and metastatic cancer.

Exploring new near-infrared fluorescent disulfide-based cyclic RGD peptide analogs for potential integrin-targeted optical imaging.

We synthesized disulfide-based cyclic RGD pentapeptides bearing a near-infrared fluorescent dye (cypate), represented by cypate-c(CRGDC) (1) for integrin-targeted optical imaging. These compounds were compared with the traditional lactam-based cyclic RGD counterpart, cypate-c(RGDfK) (2). Molecular modeling suggests that the binding affinity of 2 to integrin α(v)ß(3) is an order of magnitude higher than that of 1. This was confirmed experimentally, which further showed that substitution of Gly with Pro, Val and Tyr in 1 remarkably hampered the α(v)ß(3) binding. Interestingly, cell microscopy with A549 cells showed that 1 exhibited higher cellular staining than 2. These results indicate that factors other than receptor binding affinity to α(v)ß(3) dimeric proteins mediate cellular uptake. Consequently, 1 and its analogs may serve as valuable molecular probes for investigating the selectivity and specificity of integrin targeting by optical imaging.

Multimodal imaging of integrin receptor-positive tumors by bioluminescence, fluorescence, gamma scintigraphy, and single-photon emission computed tomography using a cyclic RGD peptide labeled with a near-infrared fluorescent dye and a radionuclide.

Integrins, particularly the alpha(v)beta(3) heterodimers, play important roles in tumor-induced angiogenesis and invasiveness. To image the expression pattern of the alpha(v)beta(3) integrin in tumors through a multimodality imaging paradigm, we prepared a cyclic RGDyK peptide analogue (LS308) bearing a tetraazamacrocycle 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and a lipophilic near-infrared (NIR) fluorescent dye cypate. The alpha(v)beta(3) integrin binding affinity and the internalization properties of LS308 mediated by the alpha(v)beta(3) integrin in 4t1luc cells were investigated by receptor binding assay and fluorescence microscopy, respectively. The in vivo distribution of (111)In-labeled LS308 in a 4t1luc tumor-bearing mouse model was studied by fluorescence, bioluminescence, planar gamma, and single-photon emission computed tomography (SPECT). The results show that LS308 has high affinity for alpha(v)beta(3) integrin and internalized preferentially via the alpha(v)beta(3) integrin-mediated endocytosis in 4t1luc cells. We also found that LS308 selectively accumulated in alpha(v)beta(3)-positve tumors in a receptor-specific manner and was visualized by the four imaging methods. Whereas the endogenous bioluminescence imaging identified the ensemble of the tumor tissue, the fluorescence and SPECT methods with the exogenous contrast agent LS308 reported the local expression of alpha(v)beta(3) integrin. Thus, the multimodal imaging approach could provide important complementary diagnostic information for monitoring the efficacy of new antiangiogenic drugs.

Novel near-infrared fluorescent integrin-targeted DFO analogue.

Desferrioxamine (DFO), a siderophore initially isolated from Streptomyces pilosus, possesses extraordinary metal binding properties with wide biomedical applications that include chelation therapy, nuclear imaging, and antiproliferation. In this work, we prepared a novel multifunctional agent consisting of (i) a near-infrared (NIR) fluorescent probe-cypate; (ii) an integrin alpha vbeta3 receptor (ABIR)-avid cyclic RGD peptide, and (iii) a DFO moiety, DFO-cypate-cyclo[RGDfK(approximately)] (1, with approximately representing the cypate conjugation site at the side chain of lysine; f is d-phenylalanine). Compound 1 and two control compounds, cypate-cyclo[RGDfK(approximately)] ( 2) and cypate-DFO ( 3), were synthesized by modular assembly of the corresponding protected RGD peptide cyclo[R(Pbf)GD(OBut)fK] and DFO on the dicarboxylic acid-containing cypate scaffold in solution. The three compounds exhibited similar UV-vis and emission spectral properties. Metal binding analysis shows that DFO as well as 1 and 3 exhibited relatively high binding affinity with Fe(III), Al(III), and Ga(III). In contrast to Ga(III), the binding of Fe to 1 and 3 quenched the fluorescence emission of cypate significantly, suggesting an efficient metal-mediated approach to perturb the spectral properties of NIR fluorescent carbocyanine probes. In vitro, 1 showed a high ABIR binding affinity (10 (-7) M) comparable to that of 2 and the reference peptide cyclo(RGDfV), indicating that both DFO and cypate motifs did not interfere significantly with the molecular recognition of the cyclic RGD motif with ABIR. Fluorescence microscopy showed that internalization of 1 and 2 in ABIR-positive A549 cells at 1 h postincubation was higher than 3 and cypate alone, demonstrating that incorporating ABIR-targeting RGD motif could improve cellular internalization of DFO analogues. The ensemble of these findings demonstrate the use of multifunctional NIR fluorescent ABIR-targeting DFO analogues to modulate the spectral properties of the NIR fluorescent probe by the chelating properties of DFO and visualize intracellular delivery of DFO by receptor-specific peptides. These features provide a strategy to explore the potential of 1 in tumor imaging and treatment as well as some molecular recognition processes mediated by metal ions.

Targeting Beta-3 integrin using a linear hexapeptide labeled with a near-infrared fluorescent molecular probe.

Biomolecules containing the RGD peptide sequence are known to bind integrins with high affinity. Studies of hexa-and hepta-peptides labeled with a near-infrared fluorescent probe (cypate) showed that rearranging the glycine in a linear RGD peptide sequence to form the GRD analogue favored the uptake of the GRD compound by alphavbeta3 integrin receptor (ABIR)-positive A549 tumor cells and tissue. The internalization of the GRD compound in A549 cells and tumor uptake in mice were inhibited by ABIR-avid peptides, suggesting its recognition by this receptor. Further studies with functional blocking antibodies and beta3 knockout cells revealed that beta3 integrin mediates the internalization of the cypate-GRD peptide. Molecular modeling studies supported preferential interaction of the probe with the beta3 subunit of integrins relative to the alphav subunit. The results demonstrate that the cypate-GRD peptide targets beta3 integrin, thereby providing a strategy to monitor drug delivery and efficacy, and physiopathologic processes mediated by this protein.

Design, synthesis, and evaluation of near infrared fluorescent multimeric RGD peptides for targeting tumors.

Molecular interactions between RGD peptides and integrins are known to mediate many biological and pathological processes. This has led to an increased interest in the development of RGD compounds with high affinity and improved selectivity for integrin receptors. In this study, we synthesized and evaluated a series of multimeric RGD compounds constructed on a dicarboxylic acid-containing near-infrared (NIR) fluorescent dye (cypate) for tumor targeting. An array of NIR fluorescent RGD compounds was prepared efficiently, including one RGD monomer (cypate-(RGD)(2)-NH(2)), two RGD dimers (cypate-(RGD)(2)-NH(2) and cypate-(RGD-NH(2))(2)), one trimer (cypate-(RGD)(3)-NH(2)), two tetramers (cypate-(RGD)(4)-NH(2) and cypate-[(RGD)(2)-NH(2)](2)), one hexamer (cypate-[(RGD)(3)-NH(2)](2)), and one octamer (cypate-[(RGD)(4)-NH(2)](2)). The binding affinity of the multimeric RGD compounds for alpha(v)beta(3) integrin receptor (ABIR) showed a remarkable increase relative to the monomer cypate-RGD-NH(2). Generally, the divalent linear arrays of the multimeric RGD units bound the ABIR with slightly higher affinity than their monovalent analogues. These results suggest that the receptor binding affinity was not only dependent on the number of RGD moieties but also on the spatial alignments of the pendant peptides. Internalization of the compounds by ABIR-positive tumor cells (A549) was monitored by NIR fluorescence microscopy. The data showed that endocytosis of the octameric RGD derivative was significantly higher by comparison to other compounds in this study. In vivo noninvasive optical imaging and biodistribution data showed that the compounds were retained in A549 tumor tissue. These results clearly demonstrated that an array of simple RGD tripeptides on a NIR fluorescent dye core can be recognized by ABIR. Optimization of the spatial alignment of the RGD moieties through careful molecular design and library construction could induce multivalent ligand-receptor interactions useful for in vivo tumor imaging and tumor-targeted therapy.

Quantitative small animal fluorescence tomography using an ultrafast gated image intensifier.

Optical approaches to small animal in vivo molecular imaging provide high sensitivity, stable non-radioactive probes, and an extensive array of functional reporting strategies. However, quantitative whole body assays remain illusive. The quantitative accuracy of optical imaging is affected by the depth of the buried target and the heterogeneity of tissue optical properties. Tomography approaches, to obtaining in-vivo optical property maps, and whole body distributions of fluorescing probes, provide a strategy for improving the quality and quantitative accuracy of small animal optical imaging. Here we present a time-resolved, charged coupled device (CCD) based system for quantitative small animal fluorescence tomography.

Whole-body fluorescence lifetime imaging of a tumor-targeted near-infrared molecular probe in mice.

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 1x10(-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.

Monomolecular multimodal fluorescence-radioisotope imaging agents.

Diagnosis of diseases by different imaging methods can provide complementary information about the functional status of diseased tissues or organs. To overcome the current difficulties in coregistering images from different imaging modalities with a high degree of accuracy, we prepared near-infrared (NIR) monomolecular multimodal imaging agents (MOMIAs) consisting of a heptamethine carbocyanine and 111In-DOTA chelate that served as antennae for optical and scintigraphic imaging, respectively. Their spectral properties clearly show that coordination of indium to MOMIA increased the fluorescence intensity of the compounds. The MOMIAs are exceptionally stable in biological media and serum up to 24 h at 37 degrees C. Biodistribution of the compounds in mice obtained by fluorescence photon and gamma-counts demonstrated a similar distribution trend of the molecular probe in different tissues, suggesting that the detected fluorescence and gamma-emissions emanated from the same source (MOMIA). At 24 h postinjection, the MOMIAs were excreted by the renal and hepatobiliary systems and the blood level of a representative MOMIA was very low, thereby reducing background noise caused by circulating molecular probes. These findings demonstrate the feasibility of preparing single molecules with the capacity to emit discernible and diagnostic fluorescent and gamma-radiations for optical and nuclear imaging of living organisms.

Synergistic effects of light-emitting probes and peptides for targeting and monitoring integrin expression.

Integrins mediate many biological processes, including tumor-induced angiogenesis and metastasis. The arginine-glycine-aspartic acid (RGD) peptide sequence is a common recognition motif by integrins in many proteins and small peptides. While evaluating a small library of RGD peptides for imaging alpha(V)beta(3) integrin (ABI)-positive tumor cell line (A549) by optical methods, we discovered that conjugating a presumably inactive linear hexapeptide GRDSPK with a near-infrared carbocyanine molecular probe (Cypate) yielded a previously undescribed bioactive ligand (Cyp-GRD) that targets ABI-positive tumors. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay with A549 cells showed that Cyp-GRD was not cytotoxic up to 100 muM in cell culture. The compound was internalized by cells, and this internalization was blocked by coincubation with a cyclic RGD peptide (cyclo[RGDfV], f is d-phenylalanine) that binds ABI with high affinity. In vivo, Cyp-GRD selectively accumulated in tumors relative to surrounding normal tissues. Blocking studies with cyclo[RGDfV] inhibited the in vivo uptake of Cyp-GRD, suggesting that both compounds target the same active site of the protein. A strong correlation between the Cyp-GRD peptide and mitochondrial NADH concentration suggests that the new molecule could also report on the metabolic status of cells ex vivo. Interestingly, neither a Cypate-labeled linear RGD peptide nor an (111)In-labeled DOTA-GRD conjugate was selectively retained in the tumor. These results clearly demonstrate the synergistic effects of Cypate and GRD peptide for molecular recognition of integrin expression and suggest the potential of using carbocyanines as optical scaffolds for designing biologically active molecules.

Multivalent carbocyanine molecular probes: synthesis and applications.

Synergistic multivalent interactions can amplify desired chemical or biological molecular recognitions. We report a new class of multicarboxylate-containing carbocyanine dye constructs for use as optical scaffolds that not only serve as fluorescent antennas but also participate in structural assembly of the multivalent molecular construct. Three generations of carboxylate-terminating multivalent near-infrared carbocyanine probes from a dicarboxylic acid precursor dye (cypate) were prepared via its imino diacetic acid derivatives. Conjugation of the probes with D-(+)-glucosamine afforded dendritic arrays of the carbohydrates on an inner NIR chromophore core. All the multicarboxylate probes and their glucosamine conjugates have similar NIR spectral properties because conjugation occurred at distal positions to the inner chromophore core, thereby providing consistent and predictable spectral properties for their biological applications. Although light-induced photodamage equally affected the precursor dye, multicarboxylate probes, and their glucosamine derivatives, we observed that octacarboxylcypate (multivalent probe) was remarkably stable in different mediums at physiologically relevant temperatures relative to cypate, especially in basic mediums. Biodistribution studies in tumor-bearing nude mice show that all the glucosamine conjugates localized in the tumor but cypate was almost exclusively retained in the liver at 24 h postinjection. The tumor uptake does not correlate with the number of glucosamine tether on the multicarboxylate probe. Overall, the triglucosamine derivative appears to offer the best balance between high tumor uptake and low retention in nontarget tissues. These results suggest that multivalent molecular beacons are useful for assessing the beneficial effects of multivalency and for optimizing the biological and chemical properties of tissue-specific molecular probes.

Time-dependent whole-body fluorescence tomography of probe bio-distributions in mice.

We present a fast scanning fluorescence optical tomography system for imaging the kinetics of probe distributions through out the whole body of small animals. Configured in a plane parallel geometry, the system scans a source laser using a galvanometer mirror pair (tauswitch~1ms) over flexible source patterns, and detects excitation and emission light using a high frame rate low noise, 5 MHz electron multiplied charge-coupled device (EMCCD) camera. Phantom studies were used to evaluate resolution, linearity, and sensitivity. Time dependent (deltat=2.2 min.) in vivo imaging of mice was performed following injections of a fluorescing probe (indocyanine green). The capability to detect differences in probe delivery route was demonstrated by comparing an intravenous injection, versus an injection into a fat pocket (retro orbital injection). Feasibility of imaging the distribution of tumor-targeted molecular probes was demonstrated by imaging a breast tumor-specific near infrared polypeptide in MDA MB 361 tumor bearing nude mice. A tomography scan, at 24 hour post injection, revealed preferential uptake in the tumor relative to surrounding tissue.

Polyvalent carbocyanine molecular beacons for molecular recognitions.

Polyvalent carboxylate-terminating near-infrared (NIR) carbocyanine molecular beacons were prepared by homologation of reactive carboxyl groups of the beacon with imino diacetic acid. Their conjugation with unprotected d-(+)-glucosamine gave dendritic arrays of the carbohydrate on an inner NIR chromophore core. In vivo evaluation of the dendritic glucosamine constructs shows enhanced uptake in proliferating tumor cells relative to surrounding normal tissue. The structural framework of these polyvalent beacons permits the amplification by synergistic effects of a variety of bioactive motifs or chemical sensors in molecular recognition interactions without dramatic change of their desirable NIR spectral properties.