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.

Since numerous tumor cells overexpress the vasoactive intestinal peptide (VIP) receptor subtype 1 (VPAC1), VIP–dye conjugates would be useful as contrast agents for in vivo imaging. However, proteolytic degradation of VIP in vivo limits their diagnostic use and highlights the need for structurally optimized VIP derivatives with improved pharmacokinetics. Here, we applied parallel nano‐synthesis of cleavable peptides on cellulose membranes to perform a complete VIP substitutional analysis. The resulting 504 different VIP–dye analogs were tested for cell binding by flow cytometry. They provided a detailed analysis of amino acid positions essential for binding to VPAC1 overexpressing cells. A generalized VIP–dye binding motif derived from the substitutional analysis results served as a reference point for further optimization. An [Arg8]‐VIP‐dye analog showed increased stability towards proteolytic degradation, good tumor‐to‐tissue contrast in mice and a longer half‐life in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

We describe the design and properties of cyanine dye-peptide conjugates synthesized on solid supports for screening assays. With this approach, we demonstrate the feasibility of including a diagnostic molecule into die spot synthesis of membrane-bound peptide libraries in order to permit screening of complete dye-peptide conjugates for their potential as fluorescent contrast agents in biomedical optical imaging. A cellulose support, which is modified with a linker permitting cleavage of the dye-peptide conjugates from the support, was prepared. The attachment of dyes to the peptides is exemplified with carboxy-substituted indotricarbocyanines, which can be covalently linked to the N-terminal amino group or a lysin in the course of the synthesis. Several dye-peptide conjugates were obtained by automated peptide synthesis on resins. Furthermore, model sequences consisting of up to 11 amino acids were synthesized on cellulose in sufficient amounts and purity, thus permitting direct testing of these compounds in cell-based assays and fluorescence microscopy. The dyes show only negligible alterations in their absorption and fluorescence properties when attached to low molecular weight peptides. In conclusion, membrane-bound peptide libraries provide a powerful tool to generate large diversities of dye-labeled peptides and to make these compounds available for biological screening assays.

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.

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.

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.

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.

Treatment of mercapto-functionalized cellulose membranes with preformed Fmoc-amino acid 3-bromopropyl esters yielded membrane-bound amino acids connected via a stable thioether and a cleavable ester bond. This synthesis strategy allows the highly parallel preparation of peptides that can be solubilized from the solid support. We apply this approach to the synthesis of novel peptide–cyanine dye conjugates which are potentially useful as fluorescent contrast agents targeted to tumor-specific receptors.

Many gastroenteropancreatic tumors express receptors for somatostatin (SST) and/or vasoactive intestinal peptide (VIP). These receptors can be used as molecular targets for the delivery of contrast agents for tumor diagnostics. We have synthesized conjugates consisting of a cyanine dye and an SST analogue or VIP for use as contrast agents in optical imaging. Receptor binding and internalization of these compounds were examined with optical methods in transfected RIN38 tumor cells expressing the SST2 receptor or a GFP-labeled VIP (VPAC1) receptor. Furthermore, biodistribution of the conjugates was examined by laser-induced fluorescence imaging in nude mice bearing SST2 or VPAC1 receptor-expressing tumors. After incubation of RIN38 SSTR2 cells in the presence of 100 nM indotricarbocyanine-SST analogue, cell-associated fluorescence increased, whereas no increase was observed when receptor-mediated endocytosis was inhibited. Indodicarbocyanine-VIP accumulated in PIN38 VPAC1 cells and co-localization with the GFP-labeled VPAC1 receptor was observed. After injection of indotricarbocyanine-SST analogue into tumor-bearing nude mice, SST2 receptor-positive tumors could be visualized for a time period from 10 min to at least 48 h. After application of indodicarbocyanine-VIP, a fluorescence signal in VIP1 receptor-expressing tumors was only detected during the first hour. We conclude that cyanine dye-labeled VIP and SST analogue are novel, targeted contrast agents for the optical imaging of tumors expressing the relevant receptor.

Annals of the New York Academy of SciencesVolume 921, Issue 1 p. 275-278 Cyanine Dye Labeled Vasoactive Intestinal Peptide and Somatostatin Analog for Optical Detection of Gastroenteropancreatic Tumors ANDREAS BECKER, Corresponding Author ANDREAS BECKER Institut für Diagnostikforschung GmbH an der FU Berlin, Spandauer Damm 130, 14050 Berlin, Germany Address for correspondence. Dr Andreas Becker, Produktmanager Pharma PULSION Medical Systems AG, Stahlgruberring 28, D-81829 Munich, Germany. Voice: +49 89-45 99 14-454; fax: +49 89-45 99 14-508. becker@PULSION.deSearch for more papers by this authorCARSTEN HESSENIUS, CARSTEN HESSENIUS Universitätsklinikum Charité, Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Augustenburger Platz 1, 13353 Berlin, GermanySearch for more papers by this authorSARAH BHARGAVA, SARAH BHARGAVA Universitätsklinikum Charité, Institut für Medizinische Immunologie, Schumannstr. 20/21, 10098 Berlin, GermanySearch for more papers by this authorCARSTEN GRÖTZINGER, CARSTEN GRÖTZINGER Universitätsklinikum Charité, Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Augustenburger Platz 1, 13353 Berlin, GermanySearch for more papers by this authorKAI LICHA, KAI LICHA Institut für Diagnostikforschung GmbH an der FU Berlin, Spandauer Damm 130, 14050 Berlin, GermanySearch for more papers by this authorJENS SCHNEIDER-MERGENER, JENS SCHNEIDER-MERGENER Universitätsklinikum Charité, Institut für Medizinische Immunologie, Schumannstr. 20/21, 10098 Berlin, GermanySearch for more papers by this authorBERTRAM WIEDENMANN, BERTRAM WIEDENMANN Universitätsklinikum Charité, Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Augustenburger Platz 1, 13353 Berlin, GermanySearch for more papers by this authorWOLFHARD SEMMLER, WOLFHARD SEMMLER Institut für Diagnostikforschung GmbH an der FU Berlin, Spandauer Damm 130, 14050 Berlin, GermanySearch for more papers by this author ANDREAS BECKER, Corresponding Author ANDREAS BECKER Institut für Diagnostikforschung GmbH an der FU Berlin, Spandauer Damm 130, 14050 Berlin, Germany Address for correspondence. Dr Andreas Becker, Produktmanager Pharma PULSION Medical Systems AG, Stahlgruberring 28, D-81829 Munich, Germany. Voice: +49 89-45 99 14-454; fax: +49 89-45 99 14-508. becker@PULSION.deSearch for more papers by this authorCARSTEN HESSENIUS, CARSTEN HESSENIUS Universitätsklinikum Charité, Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Augustenburger Platz 1, 13353 Berlin, GermanySearch for more papers by this authorSARAH BHARGAVA, SARAH BHARGAVA Universitätsklinikum Charité, Institut für Medizinische Immunologie, Schumannstr. 20/21, 10098 Berlin, GermanySearch for more papers by this authorCARSTEN GRÖTZINGER, CARSTEN GRÖTZINGER Universitätsklinikum Charité, Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Augustenburger Platz 1, 13353 Berlin, GermanySearch for more papers by this authorKAI LICHA, KAI LICHA Institut für Diagnostikforschung GmbH an der FU Berlin, Spandauer Damm 130, 14050 Berlin, GermanySearch for more papers by this authorJENS SCHNEIDER-MERGENER, JENS SCHNEIDER-MERGENER Universitätsklinikum Charité, Institut für Medizinische Immunologie, Schumannstr. 20/21, 10098 Berlin, GermanySearch for more papers by this authorBERTRAM WIEDENMANN, BERTRAM WIEDENMANN Universitätsklinikum Charité, Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Augustenburger Platz 1, 13353 Berlin, GermanySearch for more papers by this authorWOLFHARD SEMMLER, WOLFHARD SEMMLER Institut für Diagnostikforschung GmbH an der FU Berlin, Spandauer Damm 130, 14050 Berlin, GermanySearch for more papers by this author First published: 25 January 2006 https://doi.org/10.1111/j.1749-6632.2000.tb06976.xCitations: 20Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume921, Issue1VIP, PACAP, GLUCAGON, AND RELATED PEPTIDES: FOURTH INTERNATIONAL SYMPOSIUMDecember 2000Pages 275-278 RelatedInformation

In this contribution we use intravital microscopy to study the dynamics of extravasation into normal and tumor tissue of several hydrophilic cyanine dyes used as near-infrared (NIR) contrast agents. The technique provides information about the angiographic properties of the dyes and about their interaction with tumor tissue under dynamic conditions in vivo. In our previous work we demonstrated that several NIR-absorbing fluorescent dyes enable in vivo fluorescence detection of tumors in mice and rats. However, the mechanism leading to dye accumulation and enhanced fluorescence in tumors is not fully understood. Increased extravasation of dyes into tumor tissue due to pathologically altered tumor vessels may be an important factor in this process. Indocyanine green (ICG) displayed predominantly intravascular distribution and rapid elimination resulting in enhanced fluorescence signal of vessels during the first 15 min after administration only. No elevated extravasation into tumor tissue was observed with ICG. A hydrophilic indotricarbocyanine derivative with a high molecular weight displayed prolonged intravascular distribution and increased fluorescence signal of the vasculature compared to surrounding tissue for up to five hours. Rapid extravasation and accumulation in tumor areas, yielding elevated contrast of tumors up to 15 min after administration, was observed with hydrophilic, low molecular weight indotricarbocyanine derivatives.

The investigation of cyanine dyes as contrast agents in optical tumor imagin has been a focus of our recent work. We have shown that i.v. injected hydrophilic indotricarbocyanine derivatives enable tumor detection by fluorescence imagin and by frequency-domain absorption spectroscopy. Our current objective is to extend this approach by conjugating these dyes with specific biomolecules in order to enhance targetability and to introduce acid-cleavable links that enable dye release in acidic cell compartments. Accordingly, we have synthesized cyanine dyes which contain different acid-cleavable hydrazone links and which were coupled to peptides, proteins and antibodies. We have studied the release of the dyes under various pH conditions. Our results show that dye release from transferrin increased under acidic conditions, while at neutral pH the stability was higher. Additionally, we observed pH-dependent fluorescence enhancement during cleavage. Cellular fluorescence microscopy experiments indicated that intracellular trapping is possible. In conclusion, cyanine dyes bound to biomolecules by acid- cleavable bonds could act as promising optical contrast agents. Further work will include optimization of release rates by chemical modification and in vivo imaging studies.

Many tumor cells are characterized by the overexpression of highly specific transferrin receptors. Transferrin is bound to its receptor, which is followed by rapid internalization into intracellular compartments; it can thus be used for the specific delivery of coupled contrast agents in to tumor cells. The aim of our study was to investigate the tumor- specificity of transferrin-modified contrast agents for optical imaging and magnetic resonance imaging (MRI). Indotricarbocyanine, a near-IR absorbing dye, and ultrasmall superparamagnetic iron oxide particles, a contrast material for MRI, were covalently coupled to transferrin and examined in vitro and in vivo for their potential as tumor-specific contrast agents. CEll culture experiments using HT29 and A431 tumor cells showed specific uptake of transferrin- modified contrast agents by the cells at 37 degrees C, while uptake was inhibited at 4 degrees C or in the presence of an excess of unlabeled transferrin. Optical imaging of tumor- bearing nude mice after intravenous injection of transferrin-ICC resulted in pronounced tumor fluorescence. Tumor specificity was also observe in MRI, where transferrin-USPIO accumulated in SMT2A tumor-bearing rats. We conclude that transferrin-modified contrast agents for optical imaging and MRI can be specifically delivered to tumor tissue by use of transferrin receptor pathways.

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.