Synthesis, Characterization, and Biological Properties of Cyanine

The ability of these agents to target the somatostatin receptor was .... Co-targeting the tumor endothelium and P-selectin-expressing glioblastoma cel...
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Bioconjugate Chem. 2001, 12, 44−50

Synthesis, Characterization, and Biological Properties of Cyanine-Labeled Somatostatin Analogues as Receptor-Targeted Fluorescent Probes Kai Licha,*,† Carsten Hessenius,‡,§ Andreas Becker,†,§ Peter Henklein,§ Michael Bauer,† Stefan Wisniewski,† Bertram Wiedenmann,‡ and Wolfhard Semmler† Institut fu¨r Diagnostikforschung GmbH an der Freien Universita¨t Berlin, 14050 Berlin, Germany, and Charite´ Medical School of the Humboldt University Berlin, Campus Virchow Hospital, Department of Internal Medicine, Division of Hepatology and Gastroenterology, 13353 Berlin, Germany, and Humboldt University Berlin, Institute of Biochemistry, 10115 Berlin, Germany. Received April 19, 2000; Revised Manuscript Received October 15, 2000

We present the synthesis and characterization of the somatostatin receptor-specific peptide H2N-(DPhe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, which is labeled with a carboxylated indodicarboand 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 H2N-(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.

INTRODUCTION

Neuropeptides and their pharmacologically optimized analogues have a unique clinical potential as carrier molecules for diagnostic and therapeutic substances. Based on the observation that many human tumors overexpress receptors for neuropeptides (1, 2), new diagnostic methods have been developed that detect and localize tumors using radiolabeled neuropeptides (3, 4). The clinical diagnostic value of receptor scintigraphy has been demonstrated by the use of [111In]DTPA-octreotide (Pentetreotide, OctreoScan), a radiodiagnostic agent with high binding affinity for somatostatin receptors (5). To date, five different somatostatin receptor subtypes have been cloned and functionally characterized (6). These receptor subtypes mediate the many physiological effects of somatostatin, for example the regulation of hormone release (7, 8). They are present in both normal (6) and tumor tissues of the central nervous system and peripheral areas including the gastroenteropancreatic system (9, 10). In vitro studies have shown that the majority of these tumors, particularly neuroendocrine tumors, preferentially express the somatostatin receptor subtype 2 (9, 11). This represents the molecular basis for therapeutic and diagnostic applications of somatostatin analogues, such as octreotide. * To whom correspondence should be adressed. Phone: ++49-30-4681 7703. Fax: ++49-30-4681 6717. E-mail: [email protected]. † Institut fu ¨ r Diagnostikforschung GmbH an der Freien Universita¨t Berlin. ‡ Charite ´ Medical School of the Humboldt University Berlin. § These authors contributed equally to the publication.

Octreotide, a synthetic octapeptide with improved metabolic stability and high binding affinity for the somatostatin receptor subtypes 2 and 5, was first used for the symptomatic treatment of patients with neuroendocrine tumors (12). The high density of somatostatin receptor subtype 2 in neuroendocrine tumors has led to the development of octreotide scintigraphy (5). In vivo visualization of somatostatin receptor positive tumors using [111In]DTPA-octreotide, is based on the binding, internalization, and retention of the radioligand in intracellular compartments such as endosomes or lysosomes (13, 14). The emerging field of biomedical optical imaging (15) has awakened a great deal of interest in developing optical contrast agents capable of engendering diseasespecific optical signals within the tissue (16). The detection of site-specific fluorescence provides a notable alternative to radioactive imaging methods, as tissue is relatively transparent for near-infrared light (700-900 nm) (15). Cyanine dyes have proven to be promising contrast agents for the in vivo fluorescence demarcation of tumors (17, 18) and have been successfully applied as fluorescent labels for target-specific carriers (19, 20). We report on the synthesis and the photophysical and biological properties of novel fluorescently labeled somatostatin receptor-specific peptides of the sequence H2N(D-Phe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, which are labeled with a carboxylated indodicarbo- or indotricarbocyanine dye at the N-terminal amino group. The compounds represent fluorescent analogues of pentetreotide ([111In]DTPA-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]Thr(ol). Using the cyanine-labeled peptides, we intend to show that the advantages of low molecular weight

10.1021/bc000040s CCC: $20.00 © 2001 American Chemical Society Published on Web 01/17/2001

Receptor-Targeted Cyanine Dye−Peptide Conjugates

Figure 1. Chemical structures of indodicarbocyanine-peptide conjugate 1 (n ) 2) and indotricarbocyanine-peptide conjugate 2 (n ) 3).

peptides are of practical use for other diagnostic techniques and permit the development of site-directed fluorescent in vivo contrast agents. For this purpose, we have synthesized and characterized two monocarboxylated cyanine dyes of different absorption wavelengths employing a carboxylic acid group that permits covalent attachment to peptides during solid-phase preparation. We describe results from the solid-phase synthetic preparation of the two cyaninelabeled octreotide-analogue peptide conjugates 1 and 2 (Figure 1). Results from studies on cellular uptake in vitro demonstrate that low molecular weight peptides, structurally based on somatostatin, enable receptormediated intracellular delivery of optical probes. EXPERIMENTAL PROCEDURES

Synthesis of Cyanine Dyes. Chemicals and Analysis. 1-(4-Sulfobutyl)-2,3,3-trimethyl-3H-indolenin (5) and 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenin (6) were synthesized as described in the literature (21, 22). Glutaconic aldehyde dianilide hydrochloride was purchased from Lancaster Synthesis (Mu¨hlheim, Germany). All other reagents and solvents were purchased from Sigma-Aldrich Co. Reactions were monitored by reversed phase TLC (RP-18 plates) with water/methanol (1:2). Purification of the synthesized dyes was performed by reversed phase chromatography on an MPLC (Kronwald, Sinsheim, Germany) with RP-18 material (Europrep 6030 C-18, 60 Å, 30 µm, Knauer) using water/methanol as eluant. The synthesized dyes were characterized by 1H NMR and mass spectroscopy. The 1H NMR spectra were recorded in CD3OD with a Bruker AC400 (400 MHz). The mass spectra were obtained with a ZAB-E spectrometer (Fisons Scientific Equipment, Loughborough, UK). Measured mass/charge ratios (m/z) and proton shifts (δ) are listed for each compound. Bis-1,1′-(4-sulfobutyl)indodicarbocyanine-5-carboxylic Acid, Sodium Salt (7). A mixture of 5 (1 g, 3.4 mmol) and malonaldehyde-bis(phenylimine) monohydrochloride (0.78 g, 3.0 mmol) in 12 mL of acetic anhydride was stirred for 30 min at 120 °C. After cooling to room temperature, 6 (1.2 g, 3.6 mmol), sodium acetate (1.0 g, 12 mmol), 12 mL of acetic anhydride, and 5 mL of acetic acid were added. The resultant mixture was heated to 120 °C under vigorous stirring, cooled to room temperature, and poured into 150 mL of diethyl ether. The precipitate was collected by filtration and then purified by reversed phase chromatography (Europrep 60-30 C-18, 60 Å, 30 µm, Knauer; eluents water/methanol 9:1 to 1:1) yielding 1.3 g (72%) 7 as a blue lyophilisate. 1 H NMR: δ 1.75 (s, 12H, 4 × CH3), 2.0 (m, 8H, 2 × CH2CH2CH2SO3-), 2.9 (t, 4H, 2 × CH2SO3-), 4.1, 4.2 (2t,

Bioconjugate Chem., Vol. 12, No. 1, 2001 45

4H, 2 × >NCH2), 6.3, 6.45 (2d, 2H, R-H, R′-H), 6.7 (t, 1H, γ-H), 7.3 (dd, 2H, 5′-H, 6′-H), 7.4 (m, 2H, 4′-H, 7′-H), 7.5 (d, 1H, 7-H), 8.05 (s, 1H, 4-H), 8.1 (d, 1H, 6-H), 8.2, 8.3 (2dd, 2H, β-H, β′-H). FAB-MS: 715 [35, (M + Na)+], 693 [70, (M + H)+], 671 [35, (M + 2H-Na)+], 91 (100). Bis-1,1′-(4-sulfobutyl)indotricarbocyanine-5-carboxylic acid, sodium salt (8). The synthesis and product isolation were performed as described for 7 using glutaconic aldehyde dianilide hydrochloride (0.86 g, 3.0 mmol) with 5 (1 g, 3.4 mmol), 6 (1.2 g, 3.6 mmol), and sodium acetate (1.0 g, 12 mmol), yielding 1.5 g (80%) 8 as a blue lyophilisate. 1H NMR: δ 1.6, 1.65 (2s, 12H, 4 × CH3), 1.7-1.9 (m, 8H, 2 × CH2CH2CH2SO3-), 2.55 (t, 4H, 2 × CH2SO3-), 3.95, 4.2 (2 t, 4H, 2 × >NCH2), 6.25 (d, 1H, R′-H), 6.55 (dd, 1H, γ′-H), 6.65 (dd, 1H, γ-H), 6.7 (d, 1H, R-H), 7.25 (d, 1H, 7-H), 7.35, 7.45 (2t, 2H, 5′-H, 6′-H), 7.6, 7.65 (2d, 2H, 4′-H, 7′-H), 7.75 (m, 2H, δ-H, β′-H), 7.9 (d, 1H, 6-H), 7.95 (s, 1H, 4-H), 8.0 (dd, 1H, β-H). FABMS: 719 [12, (M + H)+], 697 [25, (M + 2H-Na)+], 135 (100). Synthesis of Dye-Peptide Conjugates. Chemicals, Equipment, and Analysis. Fmoc-protected amino acids were purchased from Orpegen (Heidelberg, Germany) and Nova Biochem GmbH (Bad Soden, Germany) and used with the following side-chain protecting groups: tert-butoxycarbonyl (Boc) for Lys and Trp, tert-butyl ether for Thr, and Trityl (trt) for Cys. Dimethylformamide (DMF) and acetonitrile were purchased from Proligo GmbH (Hamburg, Germany), and piperidine and diisopropyl ethylamine from Sigma-Aldrich Co. Peptide resins were obtained from Rapp Polymere GmbH (Tu¨bingen, Germany). Assembly of the protected peptide chain was carried out using the stepwise solid-phase method on an automated peptide synthesizer (433A, Perkin-Elmer GmbH, U ¨ berlingen, Germany). The mass spectra (MALDITOF) were recorded on an Compact Maldi II spectrometer (Shimadzu, Japan). Analytical HPLC analysis was performed using a Nucleosil 300 C18 column (5 µm particle size; 125 × 4.6 mm); gradient: 10% B to 100% B for 45 min [(A) 1000 mL of water, 2 mL of TFA; (B) 500 mL of acetontrile, 100 mL of water, 1 mL of TFA]. Synthesis of Dye-Peptide Conjugates 1 and 2. The peptide sequence (D-Phe)-Cys-Phe-(D-Trp)-Lys-Thr-CysThr-OH was built up by Fmoc chemistry using 2-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) as the activating agent and 20% piperidine in DMF for deprotection of the Fmoc groups. In the last step of the synthesis, 7 or 8 were coupled for 18 h on resin in DMF using HBTU, diisopropyl ethylamine (preactivation with 1.1 equiv of HBTU for 15 min). After completion, the resin was washed with DMF and methylene chloride, dried, and then treated with 10 mL of a mixture containing 90% trifluoroacetic acid (TFA), 5% water, and 5% triisopropylsilane. TFA was removed under vacuum and the crude peptide precipitated with diethyl ether. Cys-Cys cyclization was achieved by dissolving the crude peptide in dimethyl sulfoxide (DMSO)/water (15:85), adjusting the pH to 7.5, and stirring the mixture for 18 h. After completion (HPLC control) the peptide solution was lyophilized twice to remove the DMSO. The crude, cyclized peptide was purified by reversed-phase HPLC on a VYDAC C18 column (40 × 300 mm, 1520 µm, 300 Å) at a flow rate of 100 mL/min using an acetonitrile gradient of 30% B to 80% B for 50 min [(A) 1000 mL of water, 2 mL of TFA; (B) 500 mL of acetontrile, 100 mL of water, 1 mL of TFA]. The fractions were analyzed by HPLC. The purified fraction containing the desired product was collected,

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freeze-dried, and analyzed by mass spectrometry (MALDITOF). (1) Calcd 1705, found 1699; (2) calcd 1731, found 1726. Synthesis of Control Dye-Peptide Conjugates 3 and 4. The synthesis of the control peptide sequence (D-Phe)Met-Phe-(D-Trp)-Lys-Thr-Met-Thr-COOH and the conjugation of dyes 7 and 8 were performed analogously. The crude product was directly subjected to chromatographic purification as described above. MALDI-TOF. (3) Calcd 1734, found 1730; (4) calcd 1760, found 1756. Photophysical Characterization. Absorption measurements were performed with a Lambda 2 photometer (Perkin-Elmer GmbH, U ¨ berlingen, Germany). Fluorescence properties were measured in phophate-buffered saline (PBS) and bovine plasma with a SPEX Fluorolog fluorometer (Instruments S. A. GmbH, Grassbrunn, Germany) (single excitation and emission monochromator, 350 W xenon lamp, PM Hamamatsu R928) with 2 µM dye solutions in front-face mode. The solutions of the indodicarbocyanine peptides 1 and 3 were excited at 590 nm, those of the indotricarbocyanine peptide conjugates 2 and 4 at 690 nm. To determine fluorescence quantum yields (QY’s), the lamp and photomultiplier were calibrated with respect to their spectral sensitivity. The quantum yields obtained are based on the standards indocyanine green (ICG) in DMSO (Φ ) 0.13) and NK1967 in DMSO (Φ ) 0.28) (23). Cell Culture Experiments. The cellular uptake of 2 and 4 was examined by fluorescence microscopy and flow cytometry using somatostatin receptor subtype 2 transfected RIN38 tumor cells (RIN38 SSTR2 cells) (24). RIN38 cells were cultured following standard procedures (RPMI 1640 medium; Life Technologies GmbH, Eggenstein, Germany) and using 5% fetal bovine serum and 5% newborn calf serum supplemented with Geneticin (200 µg/L). In preparation for flow cytometry, 1 × 105 RIN38 SSTR2 cells were cultured on 24-well microtiter plates for 3 days. Cells were incubated in 300 µL of buffer containing 50 mM Tris/HCl, pH 7.5, 5 mM MgCl2, 1 mM CaCl2, 100 mM NaCl, 4% BSA (incubation buffer) in the presence of 100 nM cyanine dye-labeled peptides for 1 h at 37 °C. After incubation, the medium was removed and the cells were washed twice with PBS. The cells were then incubated with 200 µL of a 0.25% trypsin solution in PBS for 2 min at room temperature. The cell suspension was transferred to a 5 mL polystyrene tube (Becton Dickinson, Heidelberg, Germany) and centrifuged for 5 min at 4 °C and 400g. The supernatant was removed and the cells were resuspended in 300 µL of CellFIX (Becton Dickinson, Heidelberg, Germany). FACS analysis was performed using a FACSCalibe flow cytometer (Becton Dickinson, Heidelberg, Germany) with a red laser diode (∼635 nm) for excitation and FL3 optics for detection. Cell-associated fluorescence was recorded within a defined cell population until the event of 10 000 cells was reached. For fluorescence microscopic studies RIN38 SSTR2 cells were cultured on cover slides (22 × 22 mm). After 24 h the cover slides were transferred to a six-well macro plate (Greiner GmbH, Frickenhausen, Germany) containing 0.5 mL of incubation buffer (see above). Peptide conjugate 2 or 4 was added at a final concentration of 1 µM, and incubation was carried out for 1 h at 4 °C. After incubation, the cells were washed twice in 2 mL of cold PBS, and 0.5 mL of prewarmed incubation buffer was added. The cells were incubated at 37 °C in the absence of peptides for a further 1 and 60 min. The cells were fixed by the addition of 2 mL of 0.1% CellFIX (Becton

Licha et al.

Figure 2. Scheme showing the preparation of the monocarboxylated indodicarbo- (7) and indotricarbocyanine (8), and the strcutures of the resulting peptide conjugates 1-4.

Dickinson, Heidelberg, Germany) per well for 15 min at room temperature. Cover slides were mounted for microscopic analysis using Roti Histokit (Carl Roth GmbH, Karlsruhe, Germany). The fluorescence signal of the cells was recorded using an Axiovert 135 fluorescence microscope (Carl Zeiss Jena GmbH, Go¨ttingen, Germany) equipped with a Cy 7 filter set (Exciter, HQ 710/75; Emitter, HQ 810/90; Beam splitter, Q750LP). An AttoArc HBO 100 W microscopic illuminator (Carl Zeiss Jena GmbH, Go¨ttingen, Germany) was used as a light source for fluorescence excitation. Images were taken using a thermoelectrically cooled charge-coupled device (CCD) (Micromax, model RTE/CCD-576, Princeton Instruments Inc., Trenton, NJ) and analyzed using WinView Software version 1.6.2 (Princeton Instruments Inc., Trenton, NJ). RESULTS AND DISCUSSION

Synthesis of Cyanine Dye-Peptide Conjugates. To enable a directed and uniform labeling of peptides, we synthesized two cyanine dyes bearing a carboxylic acid group as activatable moiety for further linkage. In contrast to dyes described in the literature (25-27), the carboxylic acid group is positioned at one of the benzoic rings of the chromophore, thus resulting in nonsymmetrically substituted compounds with a benzoic acidlike element. The synthesis is easily performed starting from the indolenins 5 (21) and 6 (22), giving bis-1,1′-(4sulfobutyl)indodicarbocyanine-5-carboxylic acid (7) and bis-1,1′-(4-sulfobutyl)indotricarbocyanine-5-carboxylic acid (8) in 72 and 80% yields, respectively, after chromatographic purification. This synthesis is illustrated in Figure 2. The performance of the reactions allowed the suppression of the formation of the corresponding symmetric byproducts, which were estimated (TLC; data not shown) to be less than 20% in the crude product.

Receptor-Targeted Cyanine Dye−Peptide Conjugates

Bioconjugate Chem., Vol. 12, No. 1, 2001 47

Figure 3. Reversed-phase HPLC chromatograms of 1 (A) and 2 (B). Conditions are stated in the text. Table 1. Spectral Properties of Indodicarbocyanine and Indotricarbocyanines Peptide Conjugates in Comparison with the Free Dyes compda 7 (free dye) 1 3 8 (free dye) 2 4

λmax,abs λmax,em Stokes shift  (nm) (nm) (nm) (L mol-1 cm-1) 647 648 649 747 748 748

673 670 675 782 780 784

26 22 26 35 32 36

255 000 135 000 178 000 190 000 110 000 135 000

QYb 0.30c 0.26c 0.31c 0.09d 0.10d 0.08d

Figure 4. Normalized absorbance (s) and fluorescence emission (- - -) spectra of 1 (A) and 2 (B) in PBS (pH 7.4). Concentration, 2 µM.

a Solvent PBS. b Fluorescence quantum yields based on ICG as standard (QY ) 0.13 in DMSO). c Excited at 605 nm. d Excited at 685 nm.

The somatostatin receptor-specific peptide sequence (DPhe)-Cys-Phe-(D-Trp)-Lys-Thr-Cys-Thr-OH was generated by automated solid-phase synthesis using Fmoc chemistry. For N-terminal dye attachment at the resin, 7 and 8 were readily activated using HBTU as activating agent followed by incubation with the dye reaction mixture for 18 h. TFA cleavage of protecting groups and liberation of the conjugate from the resin was possible without noticeable decomposition of the chromophore, so that conjugates 1 and 2 were assessable in good purity (above 95%) after HPLC. In Figure 3 typical chromatograms of these compounds are depicted. The control peptides, for which we expected a significantly decreased receptor affinity, were obtained by synthesizing the corresponding noncyclic peptides of the sequence (D-Phe)-Met-Phe-(D-Trp)-Lys-Thr-Met-Thr-OH with the two cysteins replaced by methionin. Dye coupling in an analogous manner led to the conjugates 3 and 4. Absorption and Fluorescence Properties. We studied the absorption and fluorescence properties of the conjugates and determined the absorption and fluorescence maxima, as well as the molar extinction and fluorescence quantum yields in comparison to the free dyes 7 and 8. Carbocyanine dyes are known to have high molar extinction coefficients with -values between 100 000 and 250 000 L mol-1 cm-1 being less affected by the chemical environment. However, fluorescence is known to be considerably quenched for certain compounds when the dye is attached to biomolecules. A general observation for cyanine dyes is that quenching of fluorescence is

Figure 5. FACS analysis of RIN38 SSTR2 cells after incubation with free dye 8, indotricarbocyanine dye-peptide conjugate 2, and the corresponding linear conjugate 4. The inserted picture represents a FACS plot of the cell population typically used. Fluorescence was recorded for 10 000 cells of the defined gate.

accompanied by deformation of the spectral absorbance profile, with the shorter-wavelength dimer shoulder being more pronounced compared with the monomer peak (28). Nevertheless, the absorption and fluorescence emission characteristics of the synthesized dye-peptide conjugates are similar to those of the free dyes, as is apparent from the data in Table 1. The molar extinction coefficients of the conjugates exhibit decreased values of around 110 000-180 000 L mol-1 cm-1 compared with those of the free dyes. Within both the indodicarbocyanine (compounds 1, 3, and 7) and indotricarbocyanine groups (compounds 2, 4, and 8) the noncyclic conjugates (3 and 4) tended to have higher extinction coefficients than the cyclic conjugates 1 and 2. In Figure 4 the absorption

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Licha et al.

Figure 6. Bright field (left row) and corresponding NIR fluorescence images (right row) of RIN38 SSTR2 cells after 1 and 60 min of incubation in the presence of 1 µM 2 (set A) and 1 µM 4 (set B) at 37 °C. The fluorescence image after 60 min at 37 °C for 4 (B) was scaled to a smaller signal range to visualize the poor cell-associated intensity.

spectra and fluorescence emission spectra measured in PBS as solvent are displayed for 1 and 2. Both the absorbance and the fluorescence spectra are typical in shape compared to known indodi- or indotricarbocyaninebased chromophores (25-27). Only the indotricarbocyanine conjugate 2 showed a more pronounced dimer shoulder (690 nm), indicating that the compound exhibits a greater tendency to form aggregates. Fluorescence quantum yields (QY) for the indodicarbocyanines 1, 3, and 7 were in the range of 30%, whereas the indotricarbocyanines 2, 4, and 8 displayed values of about 10% (Table 1). No remarkable deviations in fluorescence quantum yields were observed in either group, suggesting that the degree of aggregate formation, as supported by the absorbance profile for conjugate 2, does not influence the fluorescence properties of the dyes when conjugated to the peptide. It has to be underlined, that the extent of

aggregate formation cannot be extracted from the data described herein. Hence, further studies directly analyzing the aggregation behavior are needed. Cell Culture Studies on Cellular Uptake. To study cellular uptake by means of an overall fluorescence signal derived from a defined population of tumor cells, we used the method of flow cytometry and compared the indotricarbocyanine peptide conjugate 2 with the control conjugate 4 as well as with the free label 8. Tumor cells were incubated for 1 h at a probe concentration of 100 nM. The mean fluorescence signals obtained from the trypsinated and fixed cell population are displayed in Figure 5. The fluorescence intensity derived from 2 is about 5-fold above that from 4 and 40-fold above the signal generated by autofluorescence or by incubation with the free dye 8. The data indicate that the underlying binding mechanism of 2 to the cells is a specific receptorligand interaction.

Receptor-Targeted Cyanine Dye−Peptide Conjugates

To consolidate receptor-mediated internalization into the cell, we performed NIR microscopic studies. Figure 6 shows fluorescence microscopic images of RIN38 SSTR2 tumor cells incubated with 2 (1 µM) at two different time points as well as the corresponding bright field images. After 7 min, the fluorescence signal is distributed over the whole cell and most likely associated with the plasma membrane, whereas after 30 min of incubation the fluorescence was observed in the perinuclear region and disappeared almost completely from the membrane. The appearance of small fluorescent spots suggests that 2 is localized within intravesicular compartments. Consistent with the results from flow cytometry, only negligible perinuclear signals were detectable after 60 min of incubation at 37 °C using the control probe 4 (Figure 6B), again underlining receptor specificity of 2. The receptor affinities (IC50 values) of these optical probes remain to be investigated by an appropriate competition assay. Additionally, an important aspect of such compounds is their ability to fluorescently delineate tumors in vivo after systemic administration. This is currently being explored by fluorescence imaging in tumor-bearing animals. CONCLUSION

We have demonstrated that an octreotide-derived peptide can be conjugated to near-infrared absorbing and fluorescing cyanine dyes, and that the resulting compounds maintain their photophysical properties. Through flow cytometry and fluorescence microscopy using tumor cells we showed that the conjugates specifically bind the somatostatin receptor and are internalized into intracellular compartments. The developed peptide conjugates structurally related to [111In]DTPA-octreotide exemplify the strength of low molecular weight peptide probes as vehicles for receptortargeted optical imaging. This principle may be utilized for other clinically more relevant receptor-ligand systems, thus broadening the spectrum of applications for contrast-enhanced optical imaging. ACKNOWLEDGMENT

This work was supported by the Bundesministerium fu¨r Bildung und Forschung (Grant 0310941). We wish to thank Christiane Rheinla¨nder and Dijana Topic for their excellent technical assistance. LITERATURE CITED (1) Reubi, J. C. (1995) Neuropeptide receptors in health and disease: the molecular basis for in vivo imaging. J. Nucl. Med. 36, 1825-1835. (2) Reubi, J. C. (1997) Regulatory peptide receptors as molecular targets for cancer diagnosis and therapy. Q. J. Nucl. Med. 41, 63-70. (3) Lamberts, S. W., Bakker, W. H., Reubi, J. C., and Krenning, E. P. (1990) Somatostatin-receptor imaging in the localization of endocrine tumors. New Engl. J. Med. 323, 1246-1249. (4) Virgolini, I., Raderer, M., Kurtaran, A., Angelberger, P., Banyai, S., Yang, Q., Li, S., Banyai, M., Pidlich, J., and Niederle, B. (1994) Vasoactive intestinal peptide-receptor imaging for the localization of intestinal adenocarcinomas and endocrine tumors. New Engl. J. Med. 331, 1116-1121. (5) Krenning, E. P., Kwekkeboom, D. J., and Bakker, W. H. (1993) Somatostatin receptor scintigraphy with [111In-DTPAD-Phe1]- and [123I-Tyr3]-octreotide: The Rotterdam experience with more than 1,000 patients. Eur. J. Nucl. Med. 20, 716731.

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