carbocyanines and arginyl-glycyl-aspartic-acid

Synchronized bio-distribution of combination therapies has several merits such as synergistic effects and reduced side-effects. Co-delivery of a protein and small molecule drug using a single nanocarrier is challenging because they possess totally different characteristics. Herein, we report the development of sophisticated nanoparticles composed of lipids, calcium carbonate and RGD peptide ligands for the co-delivery of a protein and small molecule drug combination via a simple preparation method. A 'one-step' ethanol injection method was employed to prepare the highly organized nanoparticles. The nanoparticles exhibited a spherical shape with ca. 130 nm diameter, and clearly had an integrated lipid layer covering the periphery. As a ligand, an RGD-modified lipid was post-inserted into the nanoparticles, which was important to overcome the 'PEG dilemma'. The pH-sensitivity of the targeted nanoparticles contributed to the efficient intracellular co-delivery of a protein and drug combination in Colon26 tumor cells, and noticeably improved their accumulation in the tumor region of xenograft mice. Synchronized bio-distribution of the protein and drug was achieved, which was the foundation for the synergistic effects of the combination. The targeting capability of the nanoparticles along with their pH-sensitive drug release and the synchronized bio-distribution of their cargos led to the significant antitumor activity of the SOD and paclitaxel combination in mice. This study provides novel information for the design and preparation of functionalized nanoparticles for the delivery of a protein/drug combination in vivo.

Topics: Animals; Antineoplastic Agents; Calcium Carbonate; Carbocyanines; Cell Line, Tumor; Cell Membrane Permeability; Drug Liberation; Drug Therapy, Combination; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Lipids; Male; Mice; Molecular Targeted Therapy; Nanocapsules; Neoplasms, Experimental; Oligopeptides; Optical Imaging; Paclitaxel; Phosphatidylethanolamines; Serum Albumin, Bovine; Superoxide Dismutase; Surface Properties

The potential of receptor-mediated fluorescence-based image-guided surgery tracers is generally linked to the near-infrared emission profile and good-manufacturing-production availability of fluorescent dyes. Surprisingly, little is known about the critical interaction between the structural composition of the dyes and the pharmacokinetics of the tracers. In this study, a dual-modality tracer design was used to systematically and quantitatively evaluate the influence of elongation of the polymethine chain in a fluorescent cyanine dye on the imaging potential of a targeted tracer.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Fluorescent Dyes; Humans; Mice; Oligopeptides; Optical Imaging; Radioactive Tracers; Surgery, Computer-Assisted; Tissue Distribution; Tomography, Emission-Computed, Single-Photon

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies with poor patient outcomes often resulting from delayed diagnosis. Therefore, early diagnosis can lead to a better prognosis and improved outcomes. In this study, we have developed a novel conjugate complex of plectin/integrin-targeted bispecific molecular probe, termed Gd-Cy7-PTP/RGD, to be used for magnetic resonance/near-infrared imaging (MRI/NIRF) of pancreatic cancer in vivo. This bispecific molecular probe comprises four parts: Gd(III) for MRI, cyanine 7 (Cy7) for NIRF, the peptide PTP for binding to plectin-1 specifically overexpressed on the surface of PDAC cells, and the peptide RGD for targeting integrin widely expressed on pancreatic duct epithelial cells and angiogenesis. Remarkably, the combination of PTP and RGD greatly increased the targeting efficiency in vitro and in vivo compared to that of either single peptide. Moreover, such bispecific molecular probes target pancreatic neoplasms and angiogenesis simultaneously, producing a "multi-level" targeting effect confirmed by immunofluorescence testing in vitro and in vivo. Under the guidance of MRI/NIRF dual-modality imaging, NIRF-guided delineation of surgical margins during operations was successfully achieved in orthotopic pancreatic cancer. This study promotes further exploration of bispecific molecular probes for clinical application.

Topics: Animals; Benzothiazoles; Carbocyanines; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Survival; Coordination Complexes; Female; Fluorescent Dyes; Gadolinium; Humans; Infrared Rays; Integrins; Magnetic Resonance Imaging; Margins of Excision; Mice, Inbred BALB C; Mice, Nude; Molecular Probes; Oligopeptides; Optical Imaging; Pancreatic Neoplasms; Plectin

Optical imaging techniques are becoming increasingly urgent for the early detection and monitoring the progression of tumor development. However, tumor vasculature imaging has so far been largely unexplored because of the lack of suitable optical probes. In this study, we demonstrated the preparation of near-infrared (NIR) fluorescent RGD peptide probes for noninvasive imaging of tumor vasculature during tumor angiogenesis. The peptide optical probes combined the advantages of NIR emission and RGD peptide, which possesses minimal biological absorption and specially targets the integrin, which highly expressed on activated tumor endothelial cells. In vivo optical imaging of nude mice bearing pancreatic tumor showed that systemically delivered NIR probes enabled us to visualize the tumors at 24 hours post-injection. In addition, we have performed in vivo toxicity study on the prepared fluorescent RGD peptide probes formulation. The blood test results and histological analysis demonstrated that no obvious toxicity was found for the mice treated with RGD peptide probes for two weeks. These studies suggest that the NIR fluorescent peptide probes can be further designed and employed for ultrasensitive fluorescence imaging of angiogenic tumor vasculature, as well as imaging of other pathophysiological processes accompanied by activation of endothelial cells.

Topics: Animals; Carbocyanines; Female; Fluorescent Dyes; Mice, Nude; Neovascularization, Pathologic; Oligopeptides; Optical Imaging; Pancreas; Pancreatic Neoplasms; Spectroscopy, Near-Infrared

Due to the shortage of established platforms/methods for multimodality probe construction, in this study, we developed a heterofunctional chelator, BaAn(Boc)Sar, from sarcophagine cage as a general platform for dual-modality probe construction.. A dual-modality probe for positron-emission tomography (PET) and fluorescence imaging was synthesized using the developed BaAn(Boc)Sar chelator. The c(RGDyK)(2) peptide (denoted as RGD(2)) and fluorescence dye Cy5.5 were conjugated with BaAn(Boc)Sar to form BaAnSar-RGD(2)-Cy5.5. Then, BaAnSar-RGD(2)-Cy5.5 was labeled with (64)Cu in ammonium acetate buffer. PET and fluorescent imaging were carried out to evaluate (64)Cu-BaAnSar-RGD(2)-Cy5.5 in nude mice bearing U87MG glioblastoma xenograft.. The BaAnSar-RGD(2)-Cy5.5 was labeled with (64)Cu very efficiently in 0.1 M NH(4)OAc buffer within 10 min at 37 °C in the yield of 86.7 ± 4.4 % (n = 3). The specific activity of (64)Cu-BaBaSar-RGD(2) was controlled at 50-200 mCi/μmol for the consideration of both PET and optical imaging. MicroPET quantification analysis shows that the U87MG tumor uptake is 6.41 ± 0.28, 6.51 ± 1.45, and 5.92 ± 1.57 %ID/g at 1, 4, and 20 h postinjection, respectively. Good correlation was obtained between the tumor to muscle ratios measured by the radioactivity and fluorescence intensity. As a proof of concept, an animal surgery study demonstrated that this dual-modality probe would greatly benefit the patients because the PET moiety could be used for tumor detection, and the fluorescent moiety would allow image-guided surgery.. Our findings demonstrated the effectiveness and feasibility of preparing dual-modality imaging probes based on the sarcophagine scaffold. The resulting PET and fluorescent imaging probe also holds a great potential for clinical translation.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Chelating Agents; Dipeptides; Female; Fluorescence; Fluorescent Dyes; Humans; Mice; Mice, Nude; Oligopeptides; Organ Specificity; Positron-Emission Tomography; Xenograft Model Antitumor Assays

Liver cancer is the fifth most common cause of cancer deaths worldwide. Noninvasive diagnosis is difficult and the disease heterogeneity reduces the accuracy of pathological assays. Improvement in diagnostic imaging of specific molecular disease markers has provided hope for accurate and early noninvasive detection of liver cancer. However, all current imaging technologies, including ultrasonography, computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging, are not specific targets for detection of liver cancer. The aim of this study was to test the feasibility of injecting a cocktail of specific molecular imaging agents to noninvasively image liver cancer. The target-specific cocktail contained agents for imaging the neovasculature (RGD peptide), matrix metalloproteinase (MMP), and glucose transport ((18)F-fluorodeoxyglucose [(18)F-FDG]). Imaging studies were performed in liver cancer cells and xenograft models. The distribution of MMP at the intracellular level was imaged by confocal microscopy. RGD, MMP, and (18)F-FDG were imaged on tumor-bearing mice using PET, CT, X-ray, and multi-wavelength optical imaging modalities. Image data demonstrated that each agent bound to a specific disease target component. The same liver cancer xenograft contained multiple disease markers. Those disease markers were heterogenetically distributed in the same tumor nodule. The molecular imaging agents had different distributions in the whole body and inside the tumor nodule. All target-specific agents yielded high tumor-to-background ratios after injection. In conclusion, target-specific molecular imaging agents can be used to study liver cancer in vitro and in vivo. Noninvasive multimodal/multi-target-specific molecular imaging agents could provide tools to simultaneously study multiple liver cancer components.

Topics: Animals; Benzenesulfonates; Carbocyanines; Cell Line, Tumor; Fluorescent Dyes; Fluorodeoxyglucose F18; Humans; Indoles; Liver Neoplasms; Male; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Multimodal Imaging; Neoplasm Transplantation; Oligopeptides; Optical Imaging; Positron-Emission Tomography; Radiopharmaceuticals; Single-Cell Analysis; Tissue Distribution; Tomography, X-Ray Computed

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)β(3)-targeting. Compared with its monovalent counterpart cypate-c(RGDfK) (2), 1 exhibited remarkable improvements in integrin α(v)β(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.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Fluorescent Dyes; Humans; Integrin alphaVbeta3; Mice; Mice, Nude; Neoplasms; Oligopeptides; Optical Imaging

The coupling between cell-cell and cell-matrix adhesion systems is known to affect the stability of the adhesive status of cells, as well as tissue cohesion. In this work, we perform quantitative assays of integrin-cadherin cross talk in controlled and reproducible conditions. This is achieved by plating cells on microprinted fibronectin patterns of different sizes, and simulating the formation of an intercellular contact with a microbead coated with E-cadherin extracellular domains and brought to the cell membrane. Using an optical trap, we measure the average rigidity modulus of the E-cadherin bead-cell contact as a function of the contact incubation time and of the cell spreading area. For a given incubation time, this rigidity modulus decreases by three orders of magnitude as the cell-matrix contact area, A, increases from 100 to 700 μm(2). In a similar way, the dynamics of formation of the bead-cell contact gets slower as this area increases. This is clear evidence for a strong negative feedback from cell-fibronectin onto cell-cell adhesive contacts, for which we discuss some possible mechanisms.

Topics: Animals; Biomechanical Phenomena; Cadherins; Carbocyanines; Cell Adhesion; Cell Communication; Egtazic Acid; Elastic Modulus; Feedback, Physiological; Fibronectins; Green Fluorescent Proteins; Integrins; Mice; Microspheres; Oligopeptides; Polylysine; Protein Binding; Recombinant Fusion Proteins

Labeling of RGD peptides with near-infrared fluorophores yields optical probes for noninvasive imaging of tumors overexpressing ανβ3 integrins. An important prerequisite for optimum detection sensitivity in vivo is strongly absorbing and highly emissive probes with a known fluorescence lifetime. The RGD-Cy5.5 optical probe was derived by coupling Cy5.5 to a cyclic arginine-glycine-aspartic acid-d-phenylalanine-lysine (RGDfK) peptide via an aminohexanoic acid spacer. Spectroscopic properties of the probe were studied in different matrices in comparison to Cy5.5. For in vivo imaging, human glioblastoma cells were subcutaneously implanted into nude mice, and in vivo fluorescence intensity and lifetime were measured. The fluorescence quantum yield and lifetime of Cy5.5 were found to be barely affected on RGD conjugation but dramatically changed in the presence of proteins. By time domain fluorescence imaging, we demonstrated specific binding of RGD-Cy5.5 to glioblastoma xenografts in nude mice. Discrimination of unspecific fluorescence by lifetime-gated analysis further enhanced the detection sensitivity of RGD-Cy5.5-derived signals. We characterized RGD-Cy5.5 as a strongly emissive and stable probe adequate for selective targeting of ανβ3 integrins. The specificity and thus the overall detection sensitivity in vivo were optimized with lifetime gating, based on the previous determination of the probes fluorescence lifetime under application-relevant conditions.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Fluorescent Dyes; Glioblastoma; Humans; Integrin alphaVbeta3; Male; Mice; Mice, Nude; Molecular Imaging; Molecular Probes; Neoplasm Transplantation; Oligopeptides; Protein Binding; Spectroscopy, Near-Infrared; Transplantation, Heterologous

The objective of this study is to design a fluorescent imaging agent with R-Gel, one of the recombinant polymers (RCP), for renal inflammation. The R-Gel based on human type I collagen has multiple Arg-Gly-Asp (RGD) motifs which are ligands for some types of integrin receptors on the cell surface. After intravenous administration of R-Gel labeled by Cy7 of a fluorescent dye to three animal models of nephritis mousse, interstitial nephritis (by using UUO model mice), glomerulonephritis (HIGA mice), and ischemia-reperfusion injured kidney (I/R mice), the extent of fluorescent imaging at the renal inflammation was assessed. The Cy7-labeled R-Gel was accumulated in the inflammation site to a significantly greater extent than in the normal one at 24h after administration. The renal pattern of fluorescent imaging was similar to that of administration anti-Mac1 antibody. Taken together, it is conceivable that the R-Gel was targeted to macrophages infiltrated into the inflammation site of kidney.

Topics: Animals; Carbocyanines; Collagen Type I; Epithelial Cells; Fluorescence; Fluorescent Dyes; Humans; Kidney; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Nephritis; Oligopeptides; Reperfusion Injury

There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with high affinity ( approximately 10 to 30 nmol/L) to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or (64)Cu-1,4,7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) to their N termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. NIR fluorescence and microPET imaging both showed that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 hour postinjection for two high-affinity (IC(50), approximately 20 nmol/L) (64)Cu-DOTA-conjugated knottin peptides was 4.47% +/- 1.21% and 4.56% +/- 0.64% injected dose/gram (%ID/g), compared with a low-affinity knottin peptide (IC(50), approximately 0.4 mumol/L; 1.48 +/- 0.53%ID/g) and c(RGDyK) (IC(50), approximately 1 mumol/L; 2.32 +/- 0.55%ID/g), a low-affinity cyclic pentapeptide under clinical development. Furthermore, (64)Cu-DOTA-conjugated knottin peptides generated lower levels of nonspecific liver uptake ( approximately 2%ID/g) compared with c(RGDyK) ( approximately 4%ID/g) 1 hour postinjection. MicroPET imaging results were confirmed by in vivo biodistribution studies. (64)Cu-DOTA-conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Brain Neoplasms; Carbocyanines; Cell Line, Tumor; Copper Radioisotopes; Glioblastoma; Heterocyclic Compounds, 1-Ring; Humans; Integrins; Mice; Mice, Nude; Molecular Sequence Data; Oligopeptides; Peptides; Positron-Emission Tomography; Spectroscopy, Near-Infrared; Substrate Specificity; Transplantation, Heterologous

Using molecular imaging techniques, we examined interstitial alterations during postmyocardial infarction (MI) remodeling and assessed the efficacy of antiangiotensin and antimineralocorticoid intervention, alone and in combination.. The antagonists of the renin-angiotensin-aldosterone axis restrict myocardial fibrosis and cardiac remodeling after MI and contribute to improved survival. Radionuclide imaging with technetium-99m-labeled Cy5.5 RGD imaging peptide (CRIP) targets myofibroblasts and indirectly allows monitoring of the extent of collagen deposition post-MI.. CRIP was intravenously administered for gamma imaging after 4 weeks of MI in 63 Swiss-Webster mice and in 6 unmanipulated mice. Of 63 animals, 50 were treated with captopril (C), losartan (L), spironolactone (S) alone, or in combination (CL, SC, SL, and SCL), 8 mice received no treatment. Echocardiography was performed for assessment of cardiac remodeling. Hearts were characterized histopathologically for the presence of myofibroblasts and thick and thin collagen fiber deposition.. Acute MI size was similar in all groups. The quantitative CRIP percent injected dose per gram uptake was greatest in the infarct area of untreated control mice (2.30 +/- 0.14%) and decreased significantly in animals treated with 1 agent (C, L, or S; 1.71 +/- 0.35%; p = 0.0002). The addition of 2 (CL, SC, or SL 1.31 +/- 0.40%; p < 0.0001) or 3 agents (SCL; 1.16 +/- 0.26%; p < 0.0001) demonstrated further reduction in tracer uptake. The decrease in echocardiographic left ventricular function, strain and rotation parameters, as well as histologically verified deposition of thin collagen fibers, was significantly reduced in treatment groups and correlated with CRIP uptake.. Radiolabeled CRIP allows for the evaluation of the efficacy of neurohumoral antagonists after MI and reconfirms superiority of combination therapy. If proven clinically, molecular imaging of the myocardial healing process may help plan an optimal treatment for patients susceptible to heart failure.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; Carbocyanines; Cardiovascular Agents; Disease Models, Animal; Drug Therapy, Combination; Echocardiography; Fibrillar Collagens; Fibroblasts; Fibrosis; Losartan; Mice; Mineralocorticoid Receptor Antagonists; Myocardial Infarction; Myocardium; Oligopeptides; Predictive Value of Tests; Spironolactone; Technetium; Tomography, Emission-Computed, Single-Photon; Ventricular Function, Left; Ventricular Remodeling

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Carbocyanines; Cardiovascular Agents; Drug Therapy, Combination; Echocardiography; Fibrillar Collagens; Fibroblasts; Fibrosis; Heart Failure; Humans; Mineralocorticoid Receptor Antagonists; Myocardial Infarction; Myocardium; Oligopeptides; Predictive Value of Tests; Technetium; Tomography, Emission-Computed, Single-Photon; Ventricular Function, Left; Ventricular Remodeling

Optical imaging would be desirable for cancer diagnostics since it can potentially resolve relevant oncological target structures in vivo. We therefore synthesised an alpha v beta(3) targeted fluorochrome and imaged tumour xenografts with different alpha v beta(3) expression levels using both planar and tomographic optical imaging methods.. An alpha v beta(3)-targeted RGD peptide was labelled with a cyanine dye (Cy 5.5). Binding of the optical tracer was tested on M21 melanoma (n=5), HT-1080 fibrosarcoma (n=6) and MCF-7 adenocarcinoma (n=5) cells and their tumour xenografts. All optical imaging studies were performed using two-dimensional planar fluorescence reflectance imaging (FRI) technology and three-dimensional fluorescence-mediated tomography (FMT).. In vitro, the peptide-dye conjugate showed a clear binding affinity to alpha v beta(3)-positive M21 and HT-1080 cells while alpha v beta(3)-negative MCF-7 cells and pre-dosing with the free RGD peptide revealed little to no fluorescence. In vivo, tumour xenografts were clearly visualised by FRI and FMT up to 24 h post injection. FMT allowed quantification of the fluorochrome distribution in deeper tissue sections showing an average fluorochrome concentration of 417.61 +/- 105.82 nM Cy 5.5 (M21), 353.68 +/- 54.02 nM Cy 5.5 (HT-1080) and 262.83 +/- 155.36 nM Cy 5.5 (MCF-7) in the target tissue 60 min after tracer administration. Competition with the free RGD peptide resulted in a reduction in the fluorochrome concentration in M21 tumour tissue (294.35 +/- 84.27 nM).. RGD-Cy 5.5 combined with novel tomographic optical imaging methods allows non-invasive imaging of tumour-associated alpha v beta(3) expression and may thus be a promising strategy for sensitive evaluation of tumour target expression.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Cell Separation; Diagnostic Imaging; Female; Flow Cytometry; Fluorescence; Humans; Integrin alphaVbeta3; Mice; Mice, Nude; Neoplasm Transplantation; Oligopeptides; Tomography

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.

Topics: Animals; Carbocyanines; Cell Line; Drug Design; Fluorescent Dyes; Fluorometry; Humans; Integrin alphaVbeta3; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Oligopeptides; Structure-Activity Relationship; Tissue Distribution; Transplantation, Heterologous

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.

Topics: Animals; Carbocyanines; Fluorometry; Integrins; Ligands; Mice; Mice, Nude; Microscopy, Fluorescence; Molecular Probe Techniques; Neoplasms; Oligopeptides; Peptides, Cyclic; Snake Venoms; Spectrophotometry; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

Noninvasive visualization of cell adhesion molecule alpha(v)beta(3) integrin expression in vivo has been well studied by using the radionuclide imaging modalities in various preclinical tumor models. A literature survey indicated no previous use of cyanine dyes as contrast agents for in vivo optical detection of tumor integrin. Herein, we report the integrin receptor specificity of novel peptide-dye conjugate arginine-glycine-aspartic acid (RGD)-Cy5.5 as a contrast agent in vitro, in vivo, and ex vivo. The RGD-Cy5.5 exhibited intermediate affinity for alpha(v)beta(3) integrin (IC(50) = 58.1 +/- 5.6 nmol/L). The conjugate led to elevated cell-associated fluorescence on integrin-expressing tumor cells and endothelial cells and produced minimal cell fluorescence when coincubated with c(RGDyK). In vivo imaging with a prototype three-dimensional small-animal imaging system visualized subcutaneous U87MG glioblastoma xenograft with a broad range of concentrations of fluorescent probe administered via the tail vein. The intermediate dose (0.5 nmol) produces better tumor contrast than high dose (3 nmol) and low dose (0.1 nmol) during 30 minutes to 24 hours postinjection, because of partial self-inhibition of receptor-specific tumor uptake at high dose and the presence of significant amount of background fluorescence at low dose, respectively. The tumor contrast was also dependent on the mouse viewing angles. Tumor uptake of RGD-Cy5.5 was blocked by unlabeled c(RGDyK). This study suggests that the combination of the specificity of RGD peptide/integrin interaction with near-infrared fluorescence detection may be applied to noninvasive imaging of integrin expression and monitoring anti-integrin treatment efficacy providing near real-time measurements.

Topics: Animals; Brain Neoplasms; Carbocyanines; Cell Line, Tumor; Female; Fluorescence; Humans; Integrin alphaVbeta3; Mice; Mice, Nude; Neoplasm Transplantation; Oligopeptides; Transplantation, Heterologous