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1.IntroductionBiomedical optical imaging is an emerging technology that has created promising opportunities for biomedical research and medical care in the past few years.1, 2, 3 Major driving forces have been both the instrumental and physical advances in the areas of lasers, detection techniques, and image reconstruction,4, 5, 6, 7 and chemical/biotechnological progress in the design of novel fluorescent reporter systems.2, 3, 8, 9 The availability of specific fluorescent compounds reporting molecular characteristics of early diseases in vivo is crucial for the application of optical techniques as a noninvasive, easy-to-use molecular imaging modality. A host of different fluorescent organic dye entities and inorganic reporter systems have demonstrated versatile utility as biocompatible imaging probes, including targeted small molecule and peptide conjugates,10, 11, 12, 13 labeled antibodies and other proteins,14, 15, 16 and enzyme-activatable carrier systems.8, 17, 18 The ability to detect metastatically involved lymph nodes contributes decisively to the long-term success of cancer surgery. Optical methods are predestined for the imaging of superficial tissues areas. Several publications describe the use of fluorescent markers, such as organic dyes19, 20 or semiconductor quantum dots,21, 22, 23 directed toward the improved identification and mapping of lymph nodes, in particular sentinel lymph nodes during surgery, beyond the established “blue dye” procedure.24 These approaches, however, only work properly with contrast agents locally applied in the vicinity of the respective target organs and are not directed toward the characterization of disease-specific molecular events.25, 26 Particularly, the imaging of molecular expression of inflammatory processes has not been the underlying rationale of previous work. The entry of leukocytes into the lymphatic system or into inflamed tissue is mediated by cell adhesion events involving leukocytes and endothelial cells located at the wall of blood vessels. The constitutive recirculation of lymphocytes into peripheral lymph nodes—a process known as homing—requires the interaction between L-selectin, an adhesion molecule expressed exclusively on leukocytes, and glycoproteins found on the luminal side of specialized high endothelial venules (HEV) in lymph nodes.27, 28, 29, 30, 31 These glycoproteins are recognized by the immunoglobulin M (IgM) monoclonal antibody MECA-79.27, 31, 32 The contrast agent described here employs this monoclonal antibody coupled to a near-IR fluorescent cyanine dye. As the target of the antibody is found on the luminal side of the endothelial lining of lymph node blood vessels, it can easily reach its target without penetrating the surrounding tissue. Thus, the underlying targeting principle omits the need to design agents combining target affinity with the aspect of tissue penetration, which is often a limiting issue for the use of antibodies. The diagnostic potential of MECA-79 has been studied for target-specific ultrasound imaging of lymph nodes using air-filled microparticles,33 which are too big in diameter to extravasate from the blood vessel into interstitial compartments. We describe the suitability of the MECA-79 antibody as a vehicle for optical molecular imaging by studying the targeting and biodistrubution properties and the feasibility to detect vascular glycoprotein expression of lymph nodes with fluorescence imaging. 2.Experimental2.1.Materials and PreparationThe near-IR fluorescent dye used in this study was bis-1, -(4-sulfobutyl)-indotricarbocyanine-5-carboxylic acid N-hydroxysuccinimidyl ester, sodium salt. The synthesis of this compound was described previously in detail.34, 35 MECA-79 and control antibody R4-22 (both in TRIS buffer, pH 8.0, NaCl) were purchased from Pharmingen International (Heidelberg, Germany). Both antibodies are IgM-type antibodies of approximately molecular weight. For labeling with near-IR cyanine dyes, both antibodies were transferred into carbonate buffer and concentrated to (ultracentrifugation). Labeling of antibodies was achieved as follows: antibody in carbonate buffer was incubated with a 30-fold molar excess of dye (freshly prepared stock solution dye in distilled water) for at room temperature. Unreacted dye was removed by gel filtration (NAP10 column, elution with phosphate-buffered saline, pH 7.4). Radiolabeling of MECA-79 was performed with chloramin- -sodium iodide according to standard procedures.36 Briefly, antibody was treated with or and chloramin T for at room temperature, followed by and cold NaI to stop the reaction. Purification was achieved by filtration on PD10 columns (Pharmacia Biotech) using phosphate-buffered saline (PBS). Immunohistochemistry using biotinylated MECA-79 was conducted as described in Ref. 33. 2.2.Photophysical CharacterizationAbsorption spectra were measured using a Lambda-2 photometer (Perkin-Elmer GmbH, Überlingen, Germany). Dye-to-protein ratios were calculated from the absorption spectra as described in Ref. 35, assuming an OD of 1.4 for a concentration of of antibodies. Fluorescence emission spectra of aqueous dye solutions were recorded in the front-face mode using a SPEX Fluorolog fluorometer (Instruments S.A. GmbH, Grasbrunn, Germany), consisting of single excitation and emission monochromators, a xenon lamp, and a photomultiplier (R928, Hamamatsu Deutschland GmbH, Herrsching, Germany). For fluorescence quantum yield calculations, a lamp and photomultiplier were calibrated for their spectral sensitivity.35 The quantum yields obtained are based on indocyanine green (ICG) in DMSO as standard.37 2.3.Animals, Probe Injection, and Radioactive ExperimentsThe study was performed according to the Regional Animal Research Committee (authorized by Landesamt für Arbeitsschutz, Gesundheitsschutz und technische Sicherheit Berlin). Female NMRI nude mice ( in body weight) were used for all experiments described. For imaging studies, animals were anesthetized by intraperitoneal injection of ketamin/rompun (2:1) at a dose of body weight. MECA-79 cyanine dye and control IgM cyanine dye conjugate were injected intravenously via the tail vein as a bolus at a dose of per animal ( body weight) for the in vivo fluorescence imaging and ex vivo microscopic experiments described later. For radioactive organ distribution studies, mice were injected with of -labeled antibody ( for each compound and time postinjection). Animals were sacrificed and organs were prepared at 1-, 6-, 24-, and postinjection. Radioactivity was counted by gamma scintillation, values corrected for half-life decay, and expressed as percentage of injected dose/g tissue. For microautoradiography, cryosections of thickness of peripheral lymph nodes were prepared, fixed with acetone, and air dried. Sections were dehydrated in PBS and incubated with -labeled MECA-79 antibody for and washed with PBS. Subsequently, sections were dipped into liquid film emulsion (Amersham Biosciences, Freiburg, Germany) under dark room conditions. Following exposure of approximately , liquid file coated sections were developed according to the suppliers protocol and examined with light microscopy. 2.4.In-Vivo Fluorescence Imaging and Ex Vivo Macroscopic ImagingFluorescence images were obtained using an imaging setup in reflection geometry. The output pulses (pulse duration: , wavelength: , repetition rate: ) of an optical parametric oscillator (OPO, Coherent GmbH, Dieburg, Germany) pumped by the third harmonic of a Nd:YAG laser (Infinity, Coherent GmbH, Dieburg, Germany) were used to excite the dye. The laser beam was coupled into a fiber, which was bent for mode scrambling. This fiber was placed near the objective at a defined distance from the field of view so that a circular area of about in diameter was nearly homogeneously illuminated. Fluorescence images were recorded by employing an intensified CCD camera (Spectroscopy Instruments GmbH, Grasbrunn, Germany) equipped with optical long-pass filters to detect wavelengths between 800 and . The cutoff at wavelengths above is caused by a sensitivity drop of the detector. The intensifier was opened by an electrical pulse (duration ) synchronized to the laser pulse and supplied by a high voltage (HV) generator (Avtech Electrosystems Limited, Ottawa, Canada). In this way, ambient light was suppressed in fluorescence images. For detailed monitoring of dye uptake immediately after injection, 250 images were recorded in (one image in ), followed by imaging acquisitions at 1-, 2-, 3-, 24-, and postinjection. Lymph node tissues and cryosections ( slices) were prepared from animals that were sacrificed after injection of MECA-79 cyanine dye conjugate. Highly magnified images of tissue sections were taken with the setup described earlier using intermediate rings and a macrolens. 3.Results and Discussion3.1.Radio-Iodination, Organ Distribution Studies, and MicroautoradiographyThe ability of MECA-79 to act as a target-specific vehicle in vivo was evaluated first by radiodiagnostic techniques. For this purpose, MECA-79 was radio-iodiniated with sodium iodide using a standard method. The resulting product was subjected to a simple test to prove its target binding properties by incubating cryosections, which were prepared from peripheric lymph nodes of mice, with -MECA-79. A microautoradiographic analysis after fixation with a photoemulsion showed the typical pattern of HEVs in peripheral lymph nodes (Fig. 1 ), which corresponds to the immunohistochemical appearance already published (Ref. 33). Hence, MECA-79 is capable of binding to its target and is expected to accumulate at the target site after intravenous injection into animals. We furthermore studied the pharmacokinetic behavior of MECA-79 and quantified its organ concentrations in mice in comparison to an isotype control antibody of identical molecular weight (R4-22), both radio-iodinated with . The results depicted in Fig. 2 clearly show a specific uptake in lymph nodes for -MECA-79, whereas the isotype control remains in the blood with no considerable accumulation in lymph nodes. With respect to the uptake in other organs, both conjugates show a similar behavior, proving the exclusive expression of the target in the HEVs of lymph nodes.32 Therefore, MECA-79 is suited as an imaging vehicle to detect glyocprotein target expression on the luminal side of HEVs, although it had to be proven that subsequent fluorescence labeling did not interfere with binding properties of the antibody. 3.2.Synthesis and Properties of Fluorescent ConjugatesMECA-79 could be readily conjugated with the indotricarbocyanine active ester by incubating the antibody with an excess of dye, according to standard labeling procedures. The selected cyanine dye has already been employed successfully in earlier work as a label for albumin and transferrin35 and for the synthesis of receptor-targeted near-IR imaging probes.34 Labeling of MECA-79 and the isotype control antibody R4-22 yielded a dye-to-protein ratio of 8 to 10 and for both resulting conjugates calculated from the absorption spectra. The absorption maxima were for both conjugates and the fluorescence maxima 771 and for MECA-79 and isotype control, respectively. The determination of the fluorescence quantum yields resulted in 0.13 and 0.14 for MECA-79 and isotype control, respectively, indicating that the dye molecules are not subject to fluorescence quenching and undergo a quantum yield increase [value for free dye reported with 0.088 (Ref. 35)]. As a consequence of the almost identical properties, both conjugates can be administered at the same doses. In Fig. 3 , the near-IR absorption and fluorescence emission spectrum for MECA-79 dye conjugate is depicted. 3.3.Fluorescence Imaging Studies and Ex Vivo Macroimaging of Tissue SectionsThe potential of MECA-79 cyanine dye conjugate to serve as a contrast agent for optical molecular imaging was investigated in nude mice in comparison to the nonbinding control conjugate. After intravenous injection of the dye solutions, the resulting contrast enhancement between lymph nodes and surrounding tissue was followed by continuous acquisition of near-IR fluorescence frames with an imaging setup in reflectance geometry. Typical fluorescence images of two mice, one injected with MECA-79 cyanine dye and the other one with isotype control conjugate, are shown in Fig. 4 . Figures 4(a) and 4(b) illustrates fluorescence images prior to application of the conjugates, Figs. 4(c) and 4(d) after IV application, and Figs. 4(e) and 4(f) at , respectively. Consistent with the uptake studies performed with a radio-labeled MECA-79 antibody, a specific binding and uptake in lymph nodes was observed as early as after injection. The ability to visualize and resolve different locations of the lymph nodes increased with time. A semiquantitative measure for imaging contrasts was obtained from intensity values determined within lymph node areas relative to adjacent areas of normal tissue (muscle). In Fig. 5 , contrast is given as and depicted for a time frame of up to after injection, demonstrating that target binding and blood clearance continuously improves the fluorescence contrast and visibility of the lymph nodes. At after injection, maximal contrast was achieved. At that time, the fluorescence intensity of muscle, used as reference tissue, was slightly higher than the autofluorescence intensity recorded initially (data not shown). The strong fluorescence observed in the liver is consistent with the hepatobiliary elimination pathway for this kind of macromolecules. The unspecific autofluorescence can be reduced with a manganese-free diet for the animals. The results presented illustrate that it is possible to discriminate lymph nodes as small as from normal tissue in a scattering environment. The injected dose of antibody corresponds to a molar dose of of dye per kg body weight (based on a dye-to-protein ratio of 10). In comparison with doses reported in other studies,12, 14, 15 an extremely high sensitivity has been achieved. A semiquantitative analysis of the fluorescence emission intensities of removed lymph nodes was performed to obtain a better estimate on the uptake in different lymph nodes and to allow a comparison of these tissues, which is not reasonable to be done in vivo, as signals are distorted due to different depth locations of the target sites within the animal. The illustration in Fig. 6 demonstrates that in principle all peripheral lymph nodes prepared from animals injected with MECA-79 cyanine dye conjugate display higher fluorescence intensities. The cervical and inguinal lymph nodes exhibit the highest signal emission (approximately 3 to 5 fold) compared to other lymph nodes. Tissues obtained after control conjugate injection are all at a measurable but similar background or nonspecific fluorescence emission level, which can be attributed to a certain remaining blood content including nonbound antibody conjugate. Target binding of MECA-79 cyanine dye was further characterized by analyzing the lymph node sections by macroimaging. In Fig. 7 , a macroimage of a -slice of a cervical lymph node generated by using a macrolens in the imaging setup described earlier is depicted. The near-IR fluorescence pattern observed reflects the typical appearance of HEVs as shown earlier by microautoradiography and immunohistochemistry (see Fig. 1). The specificity of MECA-79 cyanine dye is supported by the observation that the control conjugate does not lead to any HEV-associated fluorescence signals in the tissue sections. In summary, MECA-79 cyanine dye conjugate leads to specific uptake in peripheral lymph nodes after intravenous injection into mice, and generates persisting fluorescence contrast. Furthermore, the conjugate was identified ex vivo in tissue preparations and cryosections showing specific vascular targeting in HEVs. 4.ConclusionWe demonstrate the utility of the IgM antibody MECA-79 as optical molecular imaging probe to target HEVs in lymph nodes. Lymph node imaging is possible with high sensitivity requiring as little as per animal of this novel near-IR imaging conjugate. Targeting endothelial surface-expressed molecules, such as glycoproteins involved in leukocyte homing, deviates the need of extravascular tissue penetration of the contrast agent. As similar endothelial glycoproteins are also expressed during inflammation, the approach holds promise for the in vivo detection of areas of chronic inflammation. 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