Bjoern Riefke

In vivo characterization of cyanine dyes as contrast agents for near-infrared imaging

In this study indotricarbocyanines were investigated in vivo as near-infrared contrast agents. The known dye indocyanine green (ICG) has several disadvantages regarding its use in near-infrared imaging. ICG has a very short plasma half- life, limited tolerability and is unstable in aqueous solutions. Therefore, several indotricarbocyanine dyes, structurally related to ICG but with different hydrophilicities and physicochemical properties, were synthesized. The tolerability of synthesized dyes was tested in mice. The pharmacokinetic behavior and elimination characteristics were studied in a rat model. The in vivo imaging properties of synthesized dyes were investigated using a tunable, pulsed, solid state laser system for excitation and an intensified CCD camera for fluorescence imaging of different tumor-bearing nude mice models and mamma-carcinoma-bearing rat models. The dye-specific fluorescence exitance was followed at different times after dye administration. The results are demonstrated in comparison to indocyanine green. Synthesized hydrophilic indotricarbocyanine dyes had longer plasma half-lives and increasing renal elimination, corresponding to higher hydrophilicity. Tolerability in mice was increased up to 60- fold compared to ICG. Increased fluorescence exitance in tumors was observed for several dyes 24 h p.i. in the tumor models studied, whereas ICG showed no tumor fluorescence signal under the same conditions.

Synthesis and characterization of cyanine dyes as contrast agents for near-infrared imaging

The diagnostic potential of the near-infrared imaging method can be enhanced using fluorescent dyes as contrast agents. In the pool of fluorescent dyes (e.g. tetrapyrroles, polymethines) the cyanine dyes are the most promising ones because of their high molar absorption between 700 and 1000 nm, minimal phototoxicity and convenient synthetic availability. The potential of indocyanine green (ICG) as a contrast agent for optical mammography has been investigated. The major drawbacks of this compound are a rapid liver uptake after intravenous injection, a low fluorescence quantum yield and low stability in aqueous solutions. Our purpose was to generate indotricarbocyanine derivatives structurally related to ICG with improved photophysical and pharmacological properties. Several dyes more hydrophilic than ICG and covering a wide polarity range were synthesized. The dyes were characterized photophysically (absorption and fluorescence spectra, molar absorptivities and fluorescence quantum yields in different physiological media) and physicochemically (partition coefficients, plasma protein binding).

Synthesis and characterization of cyanine dye poly(ethylene glycol) conjugates as contrast agents for in-vivo fluorescence imaging

Cyanine dyes are promising near-IR contrast agents because of their high molar absorption between 700 and 1000 nm, minimal phototoxicity and convenient synthetic availability. It is known that the derivatization of drugs or contrast agents with polyethylene glycol residues leads to enhanced retention in tumor tissue. The purpose of this study was to generate indotricarbocyanines with improved pharmacological properties enabling in vivo fluorescence detection of tumors. Several hydrophilic indotricarbocyanine- poly(ethylene glycol) conjugates of different molecular weight were synthesized and characterized physicochemically and photophysically in order to test their applicability as near IR contrast media.

Kontrastmittel fur die optische Mammographie

SummaryNear 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.ZusammenfassungDie Nahinfrarotbildgebung (NIR-Bildgebung) nutzt Licht des Nahinfrarot (NIR) mit einem Spektralbereich von 650–1000 nm zur Gewebedifferenzierung aus. Da NIR-Licht durch körpereigene Farbstoffe (z. B. Hämoglobin) nur geringfügig absorbiert wird, werden Eindringtiefen von mehreren Zentimetern mit diffundierenden Photonen erzielt. Ein derzeit experimentell erprobtes Anwendungsgebiet dieses Verfahrens ist die optische Mammographie. In den letzten Jahren sind verschiedene Prototypen von Mammascannern mit dem Ziel konstruiert worden, die unterschiedlichen optischen Eigenschaften von Geweben bildgebend zu erfassen. Erste klinische Vorstudien an diesen Geräten haben jedoch ergeben, daß mit den angewendeten Verfahren bei der Erkennung und Differenzierung von Brusttumoren keine ausreichende Spezifität erzielt wird. Farbstoffe als potentielle Kontrastmittel könnten diesem Mangel abhelfen. Als bildgebendes Prinzip können bei der Verwendung von Farbstoffen als Kontrastmittel sowohl die Absorption als auch deren spezifisch emittierte Fluoreszenz herangezogen werden.

Tumor detection with cyanine dye poly(ethylene glycol) conjugates as contrast agents for near-infrared imaging

The influence of the molecular weight of cyanine dye- poly(ethylene glycol) (PEG) conjugates on their pharmacokinetic behavior and on the contrast between malignant and normal tissue in fluorescence images was studied. PEG conjugates with a molecular weight ranging from 1800 to 40000 g mol-1 were investigated in a rat model. A tunable, pulsed, solid-state laser system and an intensified CCD camera served to record fluorescence images of different tumor-bearing mice and rats. The time window of increased contrast between tumor and normal tissue in fluorescence images can be adjusted by the molecular weigh of PEG residues. Furthermore, we were able to demonstrate the visualization of subcutaneous blood vessels.

In-vivo characterization of tissue efflux rates of cyanine dyes by near-infrared spectroscopy

Optical mammography is a new imaging modality currently under development. A contrast agent capable of adding malignancy differentiation signatures is expected to enhance the sensitivity and specificity characteristics of the technique. We report the in vivo characterization of hydrophilic analogs of indocyanine green (ICG) that lead to enhanced tumor absorbance compared to surrounding tissue. The tumor efflux of these dyes was quantitatively studied in tumor-bearing rats by frequency-modulated near-infrared spectroscopy. We demonstrate that the half-lives of hydrophilic cyanine dyes in tumor tissue are considerably enhanced compared to ICG, thus providing a prolonged time window for diagnosis. Moreover, absorption differences between the tumor and normal tissue were observed for 3 hours after injection of the hydrophilic glucamine derivative NIR96010. In conclusion, pharmacokinetic properties as present with NIR96010 may facilitate contrast- agent-aided optical mammography.