2.1.

Materials and Preparation

The near-IR fluorescent dye used in this study was bis-1, $1^{\prime }$ -(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 $0.5\mathrm{mg}∕\mathrm{mL}$ in $50\text{-}\mathrm{mM}$ TRIS buffer, pH 8.0, $+500\text{-}\mathrm{mM}$ NaCl) were purchased from Pharmingen International (Heidelberg, Germany). Both antibodies are IgM-type antibodies of approximately $1000\text{-}\mathrm{kDa}$ molecular weight. For labeling with near-IR cyanine dyes, both antibodies were transferred into $50\text{-}\mathrm{mM}$ carbonate buffer and concentrated to $0.5\mathrm{mg}∕\mathrm{mL}$ (ultracentrifugation). Labeling of antibodies was achieved as follows: $1\text{-}\mathrm{mg}$ antibody in $1\text{-}\mathrm{ml}$ $50\text{-}\mathrm{mM}$ carbonate buffer was incubated with a 30-fold molar excess of dye (freshly prepared stock solution $1.8\mathrm{mg}∕\mathrm{mL}$ dye in distilled water) for $24\mathrm{h}$ 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- $\mathrm{T}∕{}^{123}\mathrm{I}$ -sodium iodide according to standard procedures.³⁶ Briefly, $50\text{-}\mu \mathrm{g}$ antibody $(0.5\mathrm{mg}∕\mathrm{mL})$ was treated with $2\text{-}\mu \mathrm{L}$ $\mathrm{Na}{}^{125}\mathrm{I}$ or $\mathrm{Na}{}^{125}\mathrm{I}$ $\left(200\mu \mathrm{Ci}\right)$ and $30\text{-}\mu \mathrm{L}$ chloramin T $(8\mathrm{mg}∕\mathrm{mL})$ for $2\mathrm{min.}$ at room temperature, followed by $60\text{-}\mu \mathrm{L}$ ${\mathrm{Na}}_2{\mathrm{S}}_2{\mathrm{O}}_5$ $(8\mathrm{mg}∕\mathrm{mL})$ and $0.5\text{-}\mathrm{mL}$ cold NaI $(2\mathrm{mg}∕\mathrm{mL})$ 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 Characterization

Absorption 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 $1\mathrm{mg}∕\mathrm{ml}$ of antibodies. Fluorescence emission spectra of $2\text{-}\mu \mathrm{mol}∕\mathrm{I}$ 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 $350\text{-}\mathrm{W}$ 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 $(\varphi =0.13)$ as standard.³⁷

2.3.

Animals, Probe Injection, and Radioactive Experiments

The study was performed according to the Regional Animal Research Committee (authorized by Landesamt für Arbeitsschutz, Gesundheitsschutz und technische Sicherheit Berlin). Female NMRI nude mice ( $20\text{to}25\mathrm{g}$ 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 $1\text{-}\mathrm{ml}∕\mathrm{kg}$ 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 $25\mu \mathrm{g}$ per animal ( $1\mathrm{mg}∕\mathrm{kg}$ body weight) for the in vivo fluorescence imaging and ex vivo microscopic experiments described later. For radioactive organ distribution studies, mice were injected with $2\mu \mathrm{Ci}$ of ${}^{123}\mathrm{I}$ -labeled antibody ( $n=3$ for each compound and time postinjection). Animals were sacrificed and organs were prepared at 1-, 6-, 24-, and $48\text{-}\mathrm{h}$ 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 $10\mu \mathrm{m}$ thickness of peripheral lymph nodes were prepared, fixed with acetone, and air dried. Sections were dehydrated in PBS and incubated with ${}^{125}\mathrm{I}$ -labeled MECA-79 antibody $\left(2\mu \mathrm{Ci}\right)$ for $1\mathrm{h}$ and washed with PBS. Subsequently, sections were dipped into liquid film emulsion (Amersham Biosciences, Freiburg, Germany) under dark room conditions. Following exposure of approximately $3\mathrm{days}$ , 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 Imaging

Fluorescence images were obtained using an imaging setup in reflection geometry. The output pulses (pulse duration: $3\mathrm{ns}$ , wavelength: $740\mathrm{nm}$ , repetition rate: $100\mathrm{Hz}$ ) of an optical parametric oscillator (OPO, Coherent GmbH, Dieburg, Germany) pumped by the third harmonic $\left(355\mathrm{nm}\right)$ of a Nd:YAG laser (Infinity, Coherent GmbH, Dieburg, Germany) were used to excite the dye. The laser beam was coupled into a $600\text{-}\mu \mathrm{m}$ 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 $250\mathrm{mm}$ 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 $860\mathrm{nm}$ . The cutoff at wavelengths above $860\mathrm{nm}$ is caused by a sensitivity drop of the detector. The intensifier was opened by an electrical pulse (duration $20\mathrm{ns}$ ) 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 $600\mathrm{s}$ (one image in $2.4\mathrm{s}$ ), followed by imaging acquisitions at 1-, 2-, 3-, 24-, and $48\text{-}\mathrm{h}$ postinjection.

Lymph node tissues and cryosections ( $20\text{-}\mu \mathrm{m}$ slices) were prepared from animals that were sacrificed $24\min$ 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.

3.1.

Radio-Iodination, Organ Distribution Studies, and Microautoradiography

The 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 $\left({}^{125}\mathrm{I}\right)$ 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 ${}^{125}\mathrm{I}$ -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.

Fig. 1

Microautoradiograph showing incubation of lymph node tissue (nude mice) with ${}^{125}\mathrm{I}$ -MECA-79 IgM. The dark spots indicating specific binding to HEVs in lymph nodes (left picture) corresponds to the binding pattern obtained by immunohistochemistry (right picture, Ref. 33).

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 ${}^{123}\mathrm{I}$ . The results depicted in Fig. 2 clearly show a specific uptake in lymph nodes for ${}^{123}\mathrm{I}$ -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.

Fig. 2

Organ distribution of ${}^{123}\mathrm{I}$ -IgM control antibody (upper chart) and ${}^{123}\mathrm{I}$ -MECA-79 (lower chart) in NMRI nude mice depicted as percentage of injected dose per gram of respective tissue.

3.2.

Synthesis and Properties of Fluorescent Conjugates

MECA-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 $750\mathrm{nm}$ for both conjugates and the fluorescence maxima 771 and $773\mathrm{nm}$ 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.

Fig. 3

Normalized absorption (solid line) and fluorescence spectra (dotted line) of MECA-79 cyanine dye conjugate. The spectra for the control conjugate look almost identical and are not included here.

3.3.

Fluorescence Imaging Studies and Ex Vivo Macroimaging of Tissue Sections

The 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) $10\min$ after IV application, and Figs. 4(e) and 4(f) at $24\mathrm{h}$ , 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 $10\min$ 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 $\left({\mathrm{I}}_{\text{lymph}}\text{-}{\mathrm{I}}_{\text{muscle}}\right)∕{\mathrm{I}}_{\text{muscle}}$ and depicted for a time frame of up to $150\min$ after injection, demonstrating that target binding and blood clearance continuously improves the fluorescence contrast and visibility of the lymph nodes. At $24\mathrm{h}$ 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.

Fig. 4

Fluorescence images of mice taken before $(\mathrm{A},{\mathrm{A}}^{\prime })$ and after administration of $25\mu \mathrm{g}$ ( $1\mathrm{mg}∕\mathrm{kg}$ body weight) of MECA-79 $\left({\mathrm{A}}^{\prime }\text{-}{\mathrm{C}}^{\prime }\right)$ and control conjugate (A-C) at $10\min$ $(\mathrm{B},{\mathrm{B}}^{\prime })$ and $24\mathrm{h}$ $(\mathrm{C},{\mathrm{C}}^{\prime })$ after injection, respectively. Fluorescence intensities are given in a false color scale, i.e., red-white regions indicate high fluorescence. All images are normalized to the same scale as indicated at the right side.

Fig. 5

Contrast $\left(K\right)$ between lymph node region and leg muscle defined as $K=\left({\mathrm{I}}_{\text{lymph}}\text{-}{\mathrm{I}}_{\text{muscle}}\right)∕{\mathrm{I}}_{\text{muscle}}$ with time after application of MECA-79 cyanine dye conjugate. No contrast $K=0$ could be observed for the isotype IgG under the same conditions (graph not shown).

The results presented illustrate that it is possible to discriminate lymph nodes as small as $1{\mathrm{mm}}^3$ from normal tissue in a scattering environment. The injected dose of $25\text{-}\mu \mathrm{g}$ antibody corresponds to a molar dose of $10\mathrm{nmol}$ 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.

Fig. 6

Comparison of lymph nodes taken from two animals $24\mathrm{h}$ after application of contrast agents (left to right: cervical, axilliary, mesenterial, inguinal, and popliteal lymph node). The cross sections indicate fluorescence intensities scaled identically for both contrast agents. The cervical and popliteal nodes show the highest fluorescence intensity.

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 $20\mu$ -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.

Fig. 7

Macroimages of $20\text{-}\mu \mathrm{m}$ -thick tissue slices of a cervical lymph node. $24\mathrm{h}$ after injection of MECA-79 cyanine dye and control conjugate, respectively, mice were sacrificed and cryosections of lymph node tissue were prepared. The field of view corresponds to about $10\mathrm{mm}$ .

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.