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Cyanine Dyes as Contrast Agents in Biomedical Optical Imaging
Cyanine Dyes as Contrast Agents in Biomedical Optical Imaging1 Kai Licha, PhD, Bjo¨rn Riefke, PhD, Bernd Ebert, PhD, Carsten Gro¨tzinger, PhD
RATIONALE AND OBJECTIVES As light exhibits highest penetration into living tissue in the near-infrared (NIR) region between 700 and 900 nm, tissue probing with NIR light provides the opportunity to detect tumors or other abnormalities up to several centimeters below the tissue surface (1). To gain a deeper understanding of physiological and molecular processes in vivo, an important role is increasingly attributed to the acquisition of optical signals generated by exogenously administered fluorescent probes (2– 4). Particularly, NIRabsorbing cyanine dyes are potentially suited as fluorescent contrast agents, e.g. for fluorescence-guided endoscopy or optical mammography (1). The fact, that fluorescent dyes can be detected at low concentrations and nonionizing, harmless radiation can be applied repeatedly to the patient renders this technology particularly attractive. In the area of organic dyes, the class of cyanine dyes has proven to be most promising for biomedical applications (5). Thus, the objective of our work is the synthesis of novel cyanine dyes and their characterization as fluorescent contrast agents. Following different concepts, a hydrophilic indotricarbocyanine, which is supposed to act as extracellular contrast agent similar to MR agents, was studied on the one hand. In a different approach we demonstrate that receptor-mediated intracellular tumor targeting using cyanine dye-labeled peptides, which are structurally derived from natural ligands of heptahelical recepAcad Radiol 2002; 9(suppl 2):S320 –S322 1 From Schering AG, Research Laboratories, Mu ¨ llerstrasse 178, 13342 Berlin, Germany (K.L.); Justesa Imagen SA, Madrid, Spain (B.R.); PhysikalischTechnische Bundesanstalt, Berlin, Germany (B.E.); and Universita¨tsklinikum Charite´ der Humboldt Universita¨t zu Berlin, Germany (C.G.). Address correspondence to K.L.
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tors, is a feasible way to achieve specific accumulation and high contrasts in animal tumors. One particular approach was adapted from the radiodiagnostic agent OctreoScan® and involved the use of the peptide octreotate which is known to effectively bind to the somatostatin receptor (SSTR). MATERIALS AND METHODS The synthesis of indotricabocyanine dyes and their conjugation to peptides was performed following procedures described in the literature (2,4). Purification of products was achieved by HPLC. Confocal fluorescence microscopy (Zeiss LSMS10) and flow-cytometric studies (FACScalibur, Becton Dickinson) of cells were performed with the biomolecule conjugates to examine receptor binding and cellular uptake. The imaging potential was investigated by fluorescence imaging studies with tumorbearing mice and rats using a tunable, pulsed, solid-state laser system used for excitation (selected wavelength for synthesized dyes: 740 nm) and an intensified charge-coupled device (CCD) camera for detection. A 780 nm longwave pass filter was used to cut off reflected excitation light. RESULTS Several cyanine dyes based on indodicarbo- and indotricarbocyanine structures were synthesized either with hydrophilic residues or conjugated to the SSTR-binding peptide octreotate. The products display high molar-absorption coefficients (up to 250,000 M-1 cm-1) and sufficient fluorescence quantum yields (10 – 15%) in physiological media. In the in vivo tumor imaging studies, the hydrophilic compound “Cy-glucamide” led to an initial
Academic Radiology, Vol 9, Suppl 2, 2002
CYANINE DYES
Figure 1. Time course of tumor-to-normal tissue contrast after injection of hydrophilic Cy-glucamide (2) (circles) in rats with chemically induced multiple mammary carcinoma (dose: 2 mol/kg) in comparison to rats after injection of the same dose of Indocyanine green (triangles). Corresponding fluorescence images with Cy-glucamide obtained at 1 min and 24 h after injection.
Figure 2. Time-course of tumor-to-normal tissue contrast after injection of indotricarbocyanine octreotate (4) (squares) in mice with SSTR2-receptor-expressing RIN38 tumors in comparison to a control conjugate where two cysteins in the octreotate sequence are replaced by methionine (circles) (dose: 0.02 mol/kg). Corresponding fluorescence images obtained at 6 h p.i.
tumor contrast and, in addition, showed a clear tumor demarcation in fluorescence images 24 hours after administration. The compound is enriched in tumor interstitial space mainly due to its pharmacokinetic properties, while Indocyanine green is expectedly not leading to elevated
contrast due to its rapid clearance by the liver. No direct binding or uptake by tumor cells could be found for Cyglucamide. In Figure 1 the results of experiments using chemically induced mammary carcinoma in rats are depicted.
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It was demonstrated by fluorescence microscopy and flow cytometry, that the STTR-binding cyanine dye conjugate with the short-length peptide octreotate (4) is specifically bound to surface receptors and internalized into tumor cells. Due to its high receptor affinity a 100 times lower dose of the receptor-specific probe compared to non-specific ones could be afforded in imaging experiments. Moreover, tumor-to-normal tissue contrast in fluorescence images is already enhanced immediately after administration and reaches its optimum within 2 hours. Figure 2 shows the results of mice with tumors which were induced by a SSTR2-receptor expressing pancreatic tumor cell line. CONCLUSION Following both the non-specific approach using hydrophilic extracellular derivatives and the receptor-targeted approach using dye-peptide conjugates, the diagnostic potential of cyanine dyes was demonstrated by in vivo
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animal studies with very small injected doses. We conclude that cyanine dyes are promising contrast agents for the fluorescence-based biomedical optical imaging of tumors and might be of value for many applications which are currently evolving throughout a broad spectrum of clinical disciplines (5). REFERENCES 1. Hawrysz DJ, Sevick-Muraca EM. Developments toward diagnostic breast cancer imaging using near-infrared optical measurements and fluorescent contrast agents. Neoplasia 2000; 2:388 – 417. 2. Licha K, Riefke B, Ntziachristos, Becker A, Chance B, Semmler W. Hydrophilic cyanine dye as contrast agents for near-infrared tumor imaging: synthesis, photophysical properties and spectroscopic in vivo characterization. Photochem Photobiol 2000; 72:392–398. 3. Weissleder R, Tung CH, Mahmood U, Bognanov A. In vivo imaging of tumors with protease-acvtivated near-infrared fluorescent probes. Nature Biotechnol 1999; 17:375–378. 4. Becker A, Hessenius C, Licha K, Ebert B, Sukowski U, Semmler W, Wiedenmann B, Gro¨tzinger C. Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands. Nature Biotechnol. 2001; 19:327–331. 5. Licha K. Contrast agents for optical imaging. In: Contrast Agents (Ed.: W. Krause), Springer Verlag, Heidelberg 2002 (in press).
RATIONALE AND OBJECTIVES As light exhibits highest penetration into living tissue in the near-infrared (NIR) region between 700 and 900 nm, tissue probing with NIR light provides the opportunity to detect tumors or other abnormalities up to several centimeters below the tissue surface (1). To gain a deeper understanding of physiological and molecular processes in vivo, an important role is increasingly attributed to the acquisition of optical signals generated by exogenously administered fluorescent probes (2– 4). Particularly, NIRabsorbing cyanine dyes are potentially suited as fluorescent contrast agents, e.g. for fluorescence-guided endoscopy or optical mammography (1). The fact, that fluorescent dyes can be detected at low concentrations and nonionizing, harmless radiation can be applied repeatedly to the patient renders this technology particularly attractive. In the area of organic dyes, the class of cyanine dyes has proven to be most promising for biomedical applications (5). Thus, the objective of our work is the synthesis of novel cyanine dyes and their characterization as fluorescent contrast agents. Following different concepts, a hydrophilic indotricarbocyanine, which is supposed to act as extracellular contrast agent similar to MR agents, was studied on the one hand. In a different approach we demonstrate that receptor-mediated intracellular tumor targeting using cyanine dye-labeled peptides, which are structurally derived from natural ligands of heptahelical recepAcad Radiol 2002; 9(suppl 2):S320 –S322 1 From Schering AG, Research Laboratories, Mu ¨ llerstrasse 178, 13342 Berlin, Germany (K.L.); Justesa Imagen SA, Madrid, Spain (B.R.); PhysikalischTechnische Bundesanstalt, Berlin, Germany (B.E.); and Universita¨tsklinikum Charite´ der Humboldt Universita¨t zu Berlin, Germany (C.G.). Address correspondence to K.L.
©
AUR, 2002
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tors, is a feasible way to achieve specific accumulation and high contrasts in animal tumors. One particular approach was adapted from the radiodiagnostic agent OctreoScan® and involved the use of the peptide octreotate which is known to effectively bind to the somatostatin receptor (SSTR). MATERIALS AND METHODS The synthesis of indotricabocyanine dyes and their conjugation to peptides was performed following procedures described in the literature (2,4). Purification of products was achieved by HPLC. Confocal fluorescence microscopy (Zeiss LSMS10) and flow-cytometric studies (FACScalibur, Becton Dickinson) of cells were performed with the biomolecule conjugates to examine receptor binding and cellular uptake. The imaging potential was investigated by fluorescence imaging studies with tumorbearing mice and rats using a tunable, pulsed, solid-state laser system used for excitation (selected wavelength for synthesized dyes: 740 nm) and an intensified charge-coupled device (CCD) camera for detection. A 780 nm longwave pass filter was used to cut off reflected excitation light. RESULTS Several cyanine dyes based on indodicarbo- and indotricarbocyanine structures were synthesized either with hydrophilic residues or conjugated to the SSTR-binding peptide octreotate. The products display high molar-absorption coefficients (up to 250,000 M-1 cm-1) and sufficient fluorescence quantum yields (10 – 15%) in physiological media. In the in vivo tumor imaging studies, the hydrophilic compound “Cy-glucamide” led to an initial
Academic Radiology, Vol 9, Suppl 2, 2002
CYANINE DYES
Figure 1. Time course of tumor-to-normal tissue contrast after injection of hydrophilic Cy-glucamide (2) (circles) in rats with chemically induced multiple mammary carcinoma (dose: 2 mol/kg) in comparison to rats after injection of the same dose of Indocyanine green (triangles). Corresponding fluorescence images with Cy-glucamide obtained at 1 min and 24 h after injection.
Figure 2. Time-course of tumor-to-normal tissue contrast after injection of indotricarbocyanine octreotate (4) (squares) in mice with SSTR2-receptor-expressing RIN38 tumors in comparison to a control conjugate where two cysteins in the octreotate sequence are replaced by methionine (circles) (dose: 0.02 mol/kg). Corresponding fluorescence images obtained at 6 h p.i.
tumor contrast and, in addition, showed a clear tumor demarcation in fluorescence images 24 hours after administration. The compound is enriched in tumor interstitial space mainly due to its pharmacokinetic properties, while Indocyanine green is expectedly not leading to elevated
contrast due to its rapid clearance by the liver. No direct binding or uptake by tumor cells could be found for Cyglucamide. In Figure 1 the results of experiments using chemically induced mammary carcinoma in rats are depicted.
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It was demonstrated by fluorescence microscopy and flow cytometry, that the STTR-binding cyanine dye conjugate with the short-length peptide octreotate (4) is specifically bound to surface receptors and internalized into tumor cells. Due to its high receptor affinity a 100 times lower dose of the receptor-specific probe compared to non-specific ones could be afforded in imaging experiments. Moreover, tumor-to-normal tissue contrast in fluorescence images is already enhanced immediately after administration and reaches its optimum within 2 hours. Figure 2 shows the results of mice with tumors which were induced by a SSTR2-receptor expressing pancreatic tumor cell line. CONCLUSION Following both the non-specific approach using hydrophilic extracellular derivatives and the receptor-targeted approach using dye-peptide conjugates, the diagnostic potential of cyanine dyes was demonstrated by in vivo
S322
Academic Radiology, Vol 9, Suppl 2, 2002
animal studies with very small injected doses. We conclude that cyanine dyes are promising contrast agents for the fluorescence-based biomedical optical imaging of tumors and might be of value for many applications which are currently evolving throughout a broad spectrum of clinical disciplines (5). REFERENCES 1. Hawrysz DJ, Sevick-Muraca EM. Developments toward diagnostic breast cancer imaging using near-infrared optical measurements and fluorescent contrast agents. Neoplasia 2000; 2:388 – 417. 2. Licha K, Riefke B, Ntziachristos, Becker A, Chance B, Semmler W. Hydrophilic cyanine dye as contrast agents for near-infrared tumor imaging: synthesis, photophysical properties and spectroscopic in vivo characterization. Photochem Photobiol 2000; 72:392–398. 3. Weissleder R, Tung CH, Mahmood U, Bognanov A. In vivo imaging of tumors with protease-acvtivated near-infrared fluorescent probes. Nature Biotechnol 1999; 17:375–378. 4. Becker A, Hessenius C, Licha K, Ebert B, Sukowski U, Semmler W, Wiedenmann B, Gro¨tzinger C. Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands. Nature Biotechnol. 2001; 19:327–331. 5. Licha K. Contrast agents for optical imaging. In: Contrast Agents (Ed.: W. Krause), Springer Verlag, Heidelberg 2002 (in press).