Optoacoustic imaging and staging of inflammation in a murine model of arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is one of the most frequent inflammatory diseases, causing pain and disability in the affected joints. Early diagnosis is essential for the efficiency of symptom-targeting treatments, but its diagnosis requires careful clinical, serologic, and imaging examinations, such as magnetic resonance imaging (MRI), which is both expensive and time consuming. In an effort to provide the biomedical community with a more accessible way to assess the advancement of arthritis, this study sought to investigate the use of multispectral optoacoustic tomography (MSOT) in a murine arthritis model, to visualize the extent of inflammation in vivo through an L-selectin/P-selectin-targeting contrast agent. METHODS: Mice with collagen-induced arthritis were studied as a model of RA. MSOT was performed using an L-selectin/P-selectin-targeting contrast agent, polyanionic dendritic polyglycerol sulfate (dPGS) labeled with a near-infrared (NIR) fluorophore, to increase the contrast of the arthritic joint. The signal intensity ratios between healthy legs and arthritic legs were calculated. Findings on contrast-enhanced MRI, clinical observations, the lymphocyte:granulocyte ratio, and histologic findings served as referents for comparison. RESULTS: MSOT using an inflammation-targeting contrast agent, dPGS-NIR, allowed for accurate diagnosis of inflammation in the mouse joints. In addition, use of this technique resulted in significant differentiation of the inflamed joints from the healthy joints (P = 0.023). The observed advancement of arthritis on the MSOT images was confirmed by clinical observation, blood analysis, contrast-enhanced MRI, and ex vivo histologic examinations. CONCLUSION: This study demonstrates that the combination of an inflammation-targeting contrast agent and optoacoustic tomographic imaging presents a promising means for the diagnosis of RA and the staging of arthritis-related inflammation.

Exploiting Fluorescence Lifetime Plasticity in FLIM: Target Molecule Localization in Cells and Tissues.

The mechanisms of drug-receptor interactions and the controlled delivery of drugs via biodegradable and biocompatible nanoparticulate carriers are active research fields in nanomedicine. Many clinically used drugs target G-protein coupled receptors (GPCRs) due to the fact that signaling via GPCRs is crucial in physiological and pathological processes and thus central for the function of biological systems. In this letter, a fast and reliable ratiometric fluorescence lifetime imaging microscopy (rmFLIM) approach is described to analyze the distribution of protein-ligand complexes in the cellular context. Binding of the fluorescently labeled antagonist naloxone to the G-protein coupled µ-opioid receptor is used as an example. To show the broad applicability of the rmFLIM method, we extended this approach to investigate the distribution of polymer-based nanocarriers in histological liver sections.

Optical imaging in drug discovery and diagnostic applications.

Optical imaging combines a variety of different diagnostic modalities which have shown great promise for biomedical imaging and as a tool in drug discovery. Several different principles to identify and characterize fundamental processes at the organ, tissue, cellular and molecular level have been exploited, supported by the design of novel imaging agents and biomolecular reporter systems. New optical imaging procedures will contribute considerably to the improvement of the knowledge of disease processes and the more efficient evaluation of drug effects in living laboratory animals. They may also find new diagnostic and therapeutic applications in human clinical practices. These techniques can be used in the field of molecular imaging to allow both visualization and quantification of molecular events associated with disease in a non-invasive and radiation-free manner using relatively simple equipment. The different aspects of imaging instrumentation and methods; the achievements in synthesis and evaluation of novel imaging agents and biochemical reporters; as well as the opportunities of optical imaging in drug delivery, drug discovery and imaging diagnostics will be discussed in this review article.

Near-infrared fluorescent dyes for enhanced contrast in optical mammography: phantom experiments.

Optical mammography with near-infrared (NIR) light using time-domain, frequency-domain, or continuous-wave techniques is a novel imaging modality to locate human breast tumors. By investigating excised specimens of normal and diseased mamma tissue we were able to demonstrate that differences in their scattering properties are a poor predictive parameter for normal and diseased mamma tissue. This paper describes the application of a NIR dye to improve the differentiation between breast tumors and normal tissue in a rat model. The NIR dye furnished a high tumor-to-tissue contrast ratio (6:1) in fluorescence images. Furthermore, this dye was used to develop liquid scattering phantoms with absorbing and fluorescent inhomogeneities. Using frequency-domain and time-domain instrumentation these inhomogeneities were localized at sufficient contrast by their increased absorption and fluorescence. Contrast between inhomogeneities and surrounding medium could be improved by combining fluorescence and transmittance images.

Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands.

We report here the in vivo diagnostic use of a peptide-dye conjugate consisting of a cyanine dye and the somatostatin analog octreotate as a contrast agent for optical tumor imaging. When used in whole-body in vivo imaging of mouse xenografts, indotricarbocyanine-octreotate accumulated in tumor tissue. Tumor fluorescence rapidly increased and was more than threefold higher than that of normal tissue from 3 to 24 h after application. The targeting conjugate was also specifically internalized by primary human neuroendocrine tumor cells. This imaging approach, combining the specificity of ligand/receptor interaction with near-infrared fluorescence detection, may be applied in various other fields of cancer diagnosis.

Synthesis, characterization, and biological properties of cyanine-labeled somatostatin analogues as receptor-targeted fluorescent probes.

We present the synthesis and characterization of the somatostatin receptor-specific peptide H(2)N-(D-Phe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, which is labeled with a carboxylated indodicarbo- and an indotricarbocyanine dye at the N-terminal amino group. The preparation was performed by automated solid-phase synthesis, with subsequent attachment of the cyanine dye and cleavage of the entire conjugate from the resin. The compounds display high molar absorbance and fluorescence quantum yields typical for cyanine dyes and are thus suitable receptor-targeted contrast agents for molecular optical imaging. The ability of these agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, in which the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. In contrast, the corresponding linearized derivative of the sequence H(2)N-(D-Phe)-Met-Phe-(D-Trp)-Lys-Thr-Met-Thr-OH produced only minimal cell fluorescence, hence confirming the specificity of the cyclic somatostatin analogue. Intracellular localization could be visualized by near-infrared (NIR) fluorescence microscopy. In conclusion, receptor-specific peptides are promising tools for designing site-directed optical contrast agents for use in molecular optical imaging.

Hydrophilic cyanine dyes as contrast agents for near-infrared tumor imaging: synthesis, photophysical properties and spectroscopic in vivo characterization.

We have synthesized a group of glucamine and gluosamine-substituted cyanine dyes structurally related to indocyanine green (ICG) and have characterized these compounds with regard to their potential as contrast agents for biomedical optical imaging. The compounds reported herein exhibit increased hydrophilicity and less plasma protein binding (< 50%), and are thus expected to have different pharmacokinetic properties compared with ICG. Furthermore, we measured enhanced fluorescence quantum yields (7-15%) in a physiological environment with respect to ICG. For the derivative with the highest hydrophilicity (5a) the efflux from tumor and normal tissue was monitored by intensity-modulated diffuse optical spectroscopy after intravenous injection into tumor-bearing rats. In comparison with ICG, 5a exhibited a considerably enhanced tissue-efflux half-life (73 min versus less than 10 min for ICG in tumor tissue), a two-fold higher initial tissue absorption coefficient compared to ICG, and finally, it generated an elevated tumor-to-tissue concentration gradient up to 1 h after injection. In conclusion, compounds such as 5a are promising contrast agents for optical imaging, and could facilitate highly sensitive and specific detection of breast cancer or other malignancies by utilizing mechanisms similar to contrast-enhanced magnetic resonance imaging or computerized tomography.

Macromolecular contrast agents for optical imaging of tumors: comparison of indotricarbocyanine-labeled human serum albumin and transferrin.

Macromolecules accumulate in solid tumors and can thus be used as carriers for the delivery of attached contrast agents to tumors. We report the synthesis and use of serum protein-dye conjugates consisting of transferrin (Tf) or human serum albumin (HSA) and an indotricarbocyanine (ITCC) derivative as contrast agents for the optical imaging of tumors. The compounds were characterized with respect to their photophysical properties and tested in vitro for their ability to bind to tumor cells and in vivo for their potential to delineate experimental tumors. In contrast to HAS-ITTC, Tf-ITCC showed receptor-mediated uptake by HT29 human colon cancer cells in vitro. After intravenous injection into HT29 tumor-bearing nude mice both compounds induced increased fluorescence contrast of tumors in vivo. After 24 h the contrast between tumor and normal tissue was significantly higher for Tf-ITCC than for HAS-ITCC. Dye-induced fluorescence was found to be predominantly located in perinecrotic areas of the tumor. Furthermore, Tf-ITCC produced fluorescence of viable tumor cells, whereas HAS-ITCC fluorescence was recorded along connective tissue. We conclude that ITCC-labeled Tf and HSA can serve as macromolecular contrast agents for the optical imaging of tumors, with Tf-ITCC showing higher efficiency.

Functional and structural characterization of synthetic HIV-1 Vpr that transduces cells, localizes to the nucleus, and induces G2 cell cycle arrest.

Human immunodeficiency virus (HIV) Vpr contributes to nuclear import of the viral pre-integration complex and induces G(2) cell cycle arrest. We describe the production of synthetic Vpr that permitted the first studies on the structure and folding of the full-length protein. Vpr is unstructured at neutral pH, whereas under acidic conditions or upon addition of trifluorethanol it adopts alpha-helical structures. Vpr forms dimers in aqueous trifluorethanol, whereas oligomers exist in pure water. (1)H NMR spectroscopy allows the signal assignment of N- and C-terminal amino acid residues; however, the central section of the molecule is obscured by self-association. These findings suggest that the in vivo folding of Vpr may require structure-stabilizing interacting factors such as previously described interacting cellular and viral proteins or nucleic acids. In biological studies we found that Vpr is efficiently taken up from the extracellular medium by cells in a process that occurs independent of other HIV-1 proteins and appears to be independent of cellular receptors. Following cellular uptake, Vpr is efficiently imported into the nucleus of transduced cells. Extracellular addition of Vpr induces G(2) cell cycle arrest in dividing cells. Together, these findings raise the possibility that circulating forms of Vpr observed in HIV-infected patients may exert biological effects on a broad range of host target cells.

[Contrast media for optical mammography]. / Kontrastmittel für die optische Mammographie.

Near infrared imaging is a non-invasive imaging modality which uses light of a spectral range between 650 and 1000 nm for tissue transillumination. Near infrared photons can penetrate by diffusion into tissue up to several centimeters due to low absorption by intrinsic chromophors (e.g. hemoglobine). During the last years optical mammography has become field of growing interest. Several prototypes of near infrared scanners have been developed for clinical studies in order to obtain transillumination images of the breast. The results indicated, that discrimination of tumors located up to several centimeters below the tissue surface (e.g. breast tumors) from surrounding healthy tissue did not succeed with sufficient specificity. The diagnostic potential of near infrared imaging can be enhanced using dyes as contrast agents. Specially designed cyanine dye derivatives are potential contrast agents because of their suitable optical properties and pharmacokinetic behaviour leading to an increased tumor fluorescence in animals after intravenous injection.