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Published online before print April 29, 2004, 10.1148/radiol.2313030425
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(Radiology 2004;231:667-673.)
© RSNA, 2004

Molecular Imaging

Molecular Targeting of Lymph Nodes with L-Selectin Ligand-specific US Contrast Agent: A Feasibility Study in Mice and Dogs1

Peter Hauff, DVM, Michael Reinhardt, Andreas Briel, PhD, Nils Debus, PhD and Michael Schirner, MD

1 From Schering, Research Laboratories, Müllerstrasse 178, D-13342 Berlin, Germany. Received March 19, 2003; revision requested May 27; final revision received September 2; accepted September 29. Address correspondence to P.H. (e-mail: peter.hauff@schering.de).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To evaluate the feasibility of using intravenously administered L-selectin ligand-specific polymer-stabilized air-filled microparticles (MPs) for active targeting of peripheral lymph nodes under normal conditions in animal models.

MATERIALS AND METHODS: L-selectin ligand-specific MPs and two control substances (immunoglobulin M–isotype MPs and native MPs) were each administered in three conscious mice as a single intravenous bolus injection (1.4 x 107 MPs/kg). All mice were sacrificed 30 minutes after administration. Lymph nodes (cervical, inguinal, axillary, popliteal, mesenteric), spleen (positive control), and kidney (blood pool control) were removed and examined for MP-related stimulated acoustic emission (SAE) signals by using harmonic color Doppler ultrasonography (US) in a tank containing degassed water. A second experiment was performed in six anesthetized beagle dogs by using the same MP formulation. Each of the MP formulations was administered in two anesthetized dogs as a single intravenous bolus injection (1 x 107 MPs/kg). The popliteal lymph nodes, spleen (positive control), and kidney (blood pool control) were examined in vivo with US for MP-related SAE signals 30 minutes after administration. Fisher exact test for the one-side alternative was used for mouse data analysis.

RESULTS: The lymph nodes of all mice (P = .05) and the popliteal lymph nodes of both dogs treated with L-selectin ligand-specific MPs showed clear MP-related SAE signals, whereas the lymph nodes of all mice and the popliteal lymph nodes of four dogs that received the control substances did not show any SAE signals.

CONCLUSION: Use of an intravenously administered L-selectin ligand-specific US contrast agent is feasible for active lymph node targeting in mice and dogs.

© RSNA, 2004

Index terms: Animals • Contrast media, experimental studies • Lymphatic system, US, 998.12983, 998.12988 • Molecular analysis • Ultrasound (US), experimental studies


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Molecular imaging is currently one of the most promising fields in medical research for the noninvasive assessment of physiologic and pathologic processes at the molecular level. This new direction has become possible because of the impressive progress in biotechnology (eg, the finalized human genome project, proteomics, and bioinformatics) during the last decade of the 20th century. Scientists involved with different imaging modalities have invested a lot of energy in the identification of suitable molecular targets and the preparation of specific contrast agents. The current favorite molecular targets are disease-associated markers, such as enzymes, receptors, and white blood cells. Enzyme activities can already be measured with positron emission tomography in humans for the determination of glucose phosphorylation in tumor cells (1). Furthermore, near-infrared fluorescence imaging probes were created for imaging of protease activity in tumor cells and were tested successfully in xenograft mouse models (2,3).

Up to now, none of the other imaging techniques in routine use seemed to be qualified for in vivo detection of enzyme activities. However, the targeting of disease-associated receptors has been described successfully for almost all imaging techniques in vitro and in animal models. {alpha}vß3–Targeted gadolinium perfluorocarbon nanoparticles (4) or paramagnetic polymerized liposomes (5) were used for the detection of tumor angiogenesis in VX2 tumor-bearing rabbits by using magnetic resonance (MR) imaging. In 2000, Bredow et al (6) were able to scintigraphically image the tumor neovasculature in tumor-bearing mice by targeting the transforming growth factor ß-binding receptor endoglin by using an appropriate radiolabeled antibody. The expression of inflammation-associated molecules was visualized with molecular probes by using MR imaging (7) and ultrasonography (US) (8) in mouse models.

The current literature shows that each of the available imaging technologies has the potential for use in molecular imaging. However, each of them has advantages and disadvantages. Nevertheless, the desired ideal molecular imaging probe and technique should fulfill such criteria as high specificity, fast target accumulation (allowing examination shortly after administration), high signal-to-noise ratio, high spatial resolution, low or no depth limitations, safety, ease of use, and reasonable cost.

The purpose of our study was to evaluate the feasibility of using intravenously administered L-selectin ligand-specific polymer-stabilized air-filled microparticles (MPs) for active targeting of peripheral lymph nodes under normal conditions in animal models.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation and Characterization of Target-specific MPs
Air-filled MPs were synthesized by adding monomeric butyl-2-cyanoacrylate (while stirring vigorously) to an acidic aqueous medium containing Triton X-100 (product no. 93426; Sigma-Aldrich Chemie, Munich, Germany) as surfactant, which resulted in an MP suspension. Afterward, the air-filled MPs were separated from non–gas-filled particles by means of flotation. The native MPs were treated under alkaline conditions, which created carboxylic groups on the particle surface. Streptavidin was linked to the outer particle shell by means of an 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (product no. E6383; Sigma-Aldrich Chemie) reaction between the amine of streptavidin and the carboxylic group of the MP. The quantity of coupled streptavidin was determined by means of titration with biotin–fluorescein isothiocyanate (FITC) (5[6]-[biotinamidohexanoylamido] pentylthioureidylfluorescein, product no. B8889; Sigma-Aldrich Chemie) and flow cytometry analysis by using the FACSCalibur system with the FL1-H setting (Becton & Dickinson, Heidelberg, Germany), an optical method for the measurement of particle-associated fluorescence intensity. The size distribution of the MPs before and after coupling with streptavidin was determined by means of single-particle optical counting (AccuSizer model 770; Particle Sizing Systems, Santa Barbara, Calif). The system was calibrated in the range of 0.5–500 µm by using polystyrene-certified size standards (Duke Scientific, Palo Alto, Calif). MECA-79 antibody (an immunoglobulin M [IgM] monoclonal antibody directed against the mouse CD62L ligand) (553863; BD Pharmingen, Heidelberg, Germany) and an IgM-isotype antibody (R4–22 [purified rat IgM,{kappa} isotype control], 553940; BD Pharmingen) was biotinylated with biotinamidocaproate n-hydroxysuccinimide ester (product no. B2643; Sigma-Aldrich Chemie) according to the recommendation of the manufacturer. The target specificity of streptavidin-loaded MPs was generated immediately prior to use by adding 5 µg of a corresponding biotinylated antibody to a suspension containing 1 x 107 MPs (10 minutes incubation time) (N.D., A.B.) (Fig 1).



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  		Figure 1. Schematic illustrates the mode of action of target-specific MPs. A, Preparation of target-specific MPs with the biotin-streptavidin approach. B, Target-specific MPs immediately after administration in the bloodstream, as well as when bound to the receptor. C, The situation 15-30 minutes (dose-dependent) after administration of target-specific MPs. The blood is completely cleared of MPs; hence, only targeted MPs are available for US and allow imaging with a strong signal-to-noise ratio.

 
Immunohistochemistry
Two popliteal lymph nodes obtained from each of three beagle dogs (kindly provided by the Schering pathology department) and two popliteal, axillary, and inguinal lymph nodes from each of five mice (kindly provided by the Schering animal facility) were isolated and investigated with regard to the specificity of MECA-79 antibody to the L-selectin ligand immunohistochemically. Freshly removed lymph nodes were immediately snap-frozen at –70°C. Immunohistochemistry was performed with 10-µm acetone-fixed cryosections. After inhibition of unspecific binding sites with 5% bovine serum albumin (product no. A7030; Sigma-Aldrich Chemie), sections were incubated for 1 hour with biotinylated MECA-79 in 1% bovine serum albumin (1:100). After blocking of endogenous peroxidase with H2O2 and incubation with an extravidin peroxidase conjugate (product no. E2886; Sigma-Aldrich Chemie), the peroxidase-based detection system (product no. IM0599; Immunotech, Bratislava, Slovak Republic) was employed with amino-ethylcarbazole as the chromogenic substrate. Specificity of staining was confirmed by means of employing an IgM isotype as a negative control (N.D.).

Animal Preparation
The study was performed according to a protocol approved by the Regional Animal Research Committee (authorized by Landesamt für Arbeitschutz, Gesundheitsschutz und technische Sicherheit Berlin No. A 0101/98). Nine conscious female mice from the Naval Medical Research Institute, or NMRI (outbred mice purchased from lab animal breeder DIMED Schönwalde, Germany; weight, 18–20 g) were used for the first experiment. The MPs were administered intravenously and were immediately followed by 500 µL of physiologic saline. Lymph nodes were investigated ex vivo with US. The second experiment was performed in six anesthetized beagle dogs (three males, three females; weight, 7.3–10.4 kg; breeder, Covance Research Products, Cumberland, Va [n = 3], and Morini, S. Polo d’Enza, Italy [n = 3]). The dogs were identified by means of numbers tattooed on their ears and were selected randomly from the stock available.

Prior to the start of the experiment, the animals were fasted for 24 hours but had free access to drinking water from an automatic trough. Initially, the dogs received a subcutaneous injection of a mixture comprising 0.1 mL Rompun (Provet, Lyssach, Switzerland) per kilogram (equivalent to 2 mL xylazine) and 0.2 mL l-Polamivet (Hoechst Veterinär, Unterschleissheim, Germany) per kilogram (equivalent to 0.5 mg levomethadone hydrochloride and 0.025 mg fenpipramide hydrochloride). The dogs were placed on a warm investigation board that contained controlled heating elements, and then they were intubated. Anesthetization was performed with 1.1%–3.0% enflurane inhalation in air during spontaneous respiration.

The region of both popliteal lymph nodes was shaved, and the residual hair was removed with a depilation cream (Pilca, Block Drug, Ratingen, Germany) to allow optimal conditions for US examination. A catheter cannula (18-G, Becton & Dickinson) was introduced into the cephalic vein of the left front leg and fixed with adhesive tape. Two three-way stopcocks connected by a transparent soft rubber tube filled with physiologic saline were used for the administration of the very low volume of MP suspension. The MPs were injected into the cavum of the three-way stopcock attached to the catheter cannula by means of injection through the rubber tube with use of a Hamilton microliter syringe fitted with a hypodermic needle (20-G, B. Braun Melsungen, Germany). The MP injection was followed immediately by 5 mL of physiologic saline injected through the other three-way stopcock. During the experiment, an electrocardiogram was recorded in standard lead I by means of adhesive electrodes (P.H., M.R.).

Contrast Material–enhanced US
Specific MECA-79 MPs and two control substances (IgM-isotype MPs and MPs without antibodies [native MPs]) were used. Each of them was administered in three conscious mice from the Naval Medical Research Institute (nine mice total, 3 x 3) as a single intravenous bolus injection (1.4 x 107 MPs/kg). Thirty minutes after substance administration, all mice were sacrificed. Lymph nodes (cervical, inguinal, axillary, popliteal, mesenteric), spleen (positive control), and kidney (blood pool control) were removed and examined for MP-related stimulated acoustic emission (SAE) signals by using harmonic color Doppler US (UM9, ATL, Bothell, Wash; L10–5 transducer; mechanical index of 1.5) in a tank containing degassed water (Fig 2) (P.H., M.R., N.D.).



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  		Figure 2. Photograph and graph illustrate the experimental setup used for ex vivo US investigations of mouse lymph nodes, spleens, and kidneys. The organ of one mouse in each group was sliced and placed in separate small agar slots and investigated in parallel for SAE signals by slowly moving the tank containing the organs below the fixed US transducer.

 
A second experiment was performed in six anesthetized beagle dogs by using the same MP formulations. Each of the MP formulations was administered in two anesthetized dogs (six dogs total, 3 x 2) as a single intravenous bolus injection (1 x 107 MPs/kg). The popliteal lymph nodes, spleen (positive control), and kidney (blood pool control) were examined in vivo with harmonic color Doppler US (UM9, ATL; L10–5 transducer; mechanical index of 1.5) for MP-related SAE signals 30 minutes after substance administration. Each lymph node was scanned twice. The second scan was performed immediately after the first to guarantee that the observed color pixels were from MPs and not from blood flow (P.H., M.R.).

Evaluation and Statistical Methods
A videorecording was made of the US examination and was assessed by two investigators (P.H., M.R.) by means of consensus. The investigators evaluated all organs and determined whether or not there were MP-related SAE signals.

For statistical analysis in the mouse experiment, because of the exploratory nature of the trial, the target variable "enhancement (yes or no)" was approached independently for all lymph nodes under discussion—namely, cervical, inguinal, axillary, popliteal, and mesenteric lymph nodes. The substance group was compared with each of the control groups by means of a 2 x 2 table and the Fisher exact test for the null hypothesis H0: pMECA-79 <= pcontrol versus the alternative H1: pMECA-79 > pcontrol, where p denotes the success rate in the corresponding group. All computations were performed by using SAS statistical software (SAS, Cary, NC). For the given sample size, a P value of .05 was the minimum attainable P value for the test used. All given P values are of purely descriptive character.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Target-specific MPs
The successful coupling of streptavidin with the MP shell was demonstrated by means of flow cytometry. Addition of biotin-FITC with increasing concentrations (0, 7 x 109, 3 x 1010, and 2 x 1011 biotin-FITC molecules) to 4 x 106 streptavidin MPs led to an increase in the fluorescence signal per particle (Fig 3). A saturation of the signal intensity was achieved with the amount of 3 x 1010 biotin-FITC molecules, which is equivalent to a biotin-FITC molecule to MP ratio of 7,500:1. This ratio reflects the loading capacity of streptavidin MP with the biotinylated MECA-79 and the respective biotinylated isotype antibody. With regard to the particle size distribution, no relevant effect of the coupling process was detectable. As a result, the number-weighted mean diameter, or Dn, and the volume-weighted mean diameter, or Dv, remained nearly the same before (Dn = 1.17 µm; Dv = 1.81 µm) and after (Dn = 1.17 µm; Dv = 1.86 µm) coupling. Ninety-nine percent of the total particle volume was smaller than 4.4 µm.



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  		Figure 3. Histogram presents increased fluorescence intensity of streptavidin MPs incubated with increasing concentrations of biotin-FITC measured by means of flow cytometry. Signal saturation was given above 3 x 1010 biotin-FITC molecules per 4 x 106 streptavidin MPs.

 
Immunohistochemistry
All dog and mouse lymph nodes were stained positively for MECA-79, which was localized mostly in the paracortical region of the lymph nodes. No signals were found by using the IgM isotype, which confirms the specificity of the MECA-79 signals.

Imaging of Mouse Lymph Nodes
The investigated lymph nodes (nine nodes per mouse) in all mice treated intravenously with specific MECA-79 MPs showed clear MP-based color signals (SAE signals), whereas the lymph nodes of all mice that received the control substances did not show any SAE signals (Table 1, Fig 4). Furthermore, no SAE signals were observed in any kidney in the mice in all three groups, which demonstrates that the bloodstream was completely cleared from circulating MPs at that time (negative control). On the other hand, the spleens in all mice in each group showed strong SAE signals, which proves that each mouse was treated with MPs that were phagocytosed physiologically by spleen macrophages (positive control). Hence, the observed SAE signals in the lymph nodes of mice treated with specific MECA-79 MPs must be related to their specific accumulation. Statistically, for each inspected lymph node (cervical, inguinal, axillary, popliteal, and mesenteric), the attained P values were .05 with the Fisher one-sided test of the substance group (specific MECA-79 MPs) versus the first control group of native MPs, as well as with the test of the substance group versus the second control group of IgM-isotype MPs.


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  		TABLE 1. Harmonic Color Doppler US Findings in Mouse Lymph Nodes, Spleens, and Kidneys

 


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  		Figure 4. US images show typical MP-based SAE signals in mouse 2, which was treated with MECA-79 MPs, and in the spleens of all mice, which were a positive control for MP accumulation. Mouse 1 (native MPs) and mouse 3 (isotype MPs) did not show any SAE signals. No SAE signals are visible in any kidneys (negative control), which demonstrates that circulating MPs were cleared from the blood.

 
In this experiment, the organs (lymph node, kidney, spleen) had to be examined ex vivo with US (Figs 2, 4) because the relatively large US transducer is inappropriate for in vivo lymph node imaging in mice.

Imaging of Dog Lymph Nodes
Imaging of lymph nodes in dogs could be performed in vivo because of the larger body size compared with the very small body size of mice. As presented in Table 2, both dogs that were treated intravenously with specific MECA-79 MPs showed strong SAE signals in both popliteal lymph nodes, whereas all dogs that received the control substances did not show any SAE signals in their corresponding lymph nodes. Furthermore, the kidney as a negative control was free of SAE signals, while the spleen as a positive control was completely filled with SAE signals in all six dogs. Although only two dogs were used per group in this study, the SAE signals in the popliteal lymph nodes of those dogs treated with specific MECA-79 MPs are regarded as specifically caused by both the use of relevant control substances and the control organs (kidney, spleen).


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  		TABLE 2. Harmonic Color Doppler US Findings in Dog Lymph Nodes, Spleens, and Kidneys

 
Moreover, no color signals were visible in a second scan of the respective lymph nodes (Fig 5, B) performed immediately after the first scan, which demonstrates that all MPs have been destroyed during the first scan. Furthermore, the lymph nodes of the dogs treated with specific MECA-79 MPs showed SAE signals mostly in the region of the paracortex (Fig 5, A), which corresponds with the location of the high endothelial venules, as confirmed by means of immunohistochemistry in Figure 5, C.



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  		Figure 5. US images show a dog lymph node treated with MECA-79 MPs. A, Four selected images from the first scan. B, Image from the second scan of the same lymph node, which was performed immediately after the first scan. The images in A show typical MP-based SAE signals in the paracortex, whereas no signals are visible in the second scan (B), which proves the destruction of MPs during the first scan. C, The SAE signal distribution in A corresponds to the high endothelial venule location immunohistochemically.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Targeting of US contrast agent has already been tested successfully in vitro (9–11) and in various animal models. There are two mechanisms described for in vivo targeting of US contrast agent: passive and active. Passive targeted US contrast agents are those that have been accumulated in cells of the reticuloendothelial system after administration and can be used, for instance, in liver (12) and lymph node (13) imaging. Active targeted US contrast agents have been made specific to cellular epitopes or other receptors by means of coupling of respective ligands such as antibodies, peptides, polysaccharides, or aptamers with the shell of stabilized microbubbles (14,15). They were prepared successfully and tested in animal models of inflammation (8,16), thrombosis (17–20), atherosclerosis (16,19,21), or angiogenesis (22). However, we are not aware of any literature that describes the use of specific US contrast agents in active targeting of healthy tissue or lymph nodes. This approach could be used, for example, in vivo for the depiction of physiologically up- or down-regulated receptors in certain organs during the postembryonic ontogenesis. This information could be helpful for a better understanding of the interactions between certain transmitters (eg, hormones, enzymes, or blood cells) and receptors during different steps of the life cycle.

To demonstrate the feasibility of this approach, we chose the L-selectin ligand expressed on the postcapillary high endothelial venules of peripheral lymph nodes under normal conditions (23). The high endothelial venules allow rapid and selective lymphocyte trafficking from the blood into the secondary lymphoid tissue. Those traffic processes are initiated by an interaction between the L-selectin expressed on the surface of lymphocytes and its ligand on the high endothelial venules (24). We were able to show that MECA-79 monoclonal antibody, which is directed to the mouse L-selectin ligand, was also specific to the same ligand in dog lymph nodes, as shown by means of immunohistochemistry. Hence, this antibody, which is also cross-reactive with L-selectin ligands in high endothelial venules of other species, such as hamsters, rabbits, pigs, and humans (25–28), was used for the preparation of specific MPs and active lymph node targeting in mice and dogs.

Furthermore, the MPs used in our study have an initial vascular phase (when they would be seen in the kidney), but it lasts for less than 30 minutes and is specific to the spleen and liver. This is why the spleen has been used as the positive control and the kidney as the negative control. Detection of lymph node–targeted MPs was performed with harmonic color Doppler US by inducing the SAE effect, a sensitive method for the depiction of resting MPs (12). These SAE signals are emitted by each MP above a certain sound pressure, which results in the disintegration of the MP while at the same time (29) resulting in typical color patterns on the monitor of a US device. This means that currently, these MPs can only be visualized for one imaging procedure. However, by using an MP formulation consisting of MP fractions with certain sensitivities to different US pressures, this procedure could be repeated by starting with low sound pressure and a stepwise increase for each new image. Although relevant control substances were carried by the bloodstream in this study, a blood pool control was used to ensure that no circulating MPs were still in the blood. The rapid blood clearance of MPs guarantees a strong signal-to-noise ratio for specific accumulated MPs at the target site. It is well known that MPs will be cleared from the blood by macrophages of the reticuloendothelial system, and this feature has already been used for the passive targeting of US contrast agents. We investigated the spleen for SAE signals as a second control to demonstrate that all animals received an MP formulation.

In summary, we were able to demonstrate the feasibility of active lymph node targeting in mice and dogs by using an intravenously administered L-selectin ligand-specific US contrast agent. Furthermore, the induction of the MP-based SAE effect in color Doppler US and the rapid blood clearance of circulating MPs yields a sensitive detection method and a strong signal-to-noise ratio of targeted MPs. To our knowledge, this is the first time that active targeting of a US contrast agent has been demonstrated in lymph nodes under normal conditions after intravenous administration. Passive lymph node targeting was already demonstrated after the interstitial injection of an echogenic perflubron emulsion (30), a polymeric US contrast agent (31), or the US contrast agent AF0150 (Imagent; Alliance Pharmaceutical, San Diego, Calif) (13) in animals by using gray scale or color Doppler US.

L-selectin ligand-specific US contrast agent could be a candidate for an indirect method of lymphography for the safe and less invasive US identification of lymph nodes—for example, when performing biopsy. Lymph node–targeted MPs can be detected easily with any US device that has color Doppler capabilities.


   ACKNOWLEDGMENTS
 
The authors thank Susanne Schwenke for her contribution to the statistical assessment, Volker Stickel and Michael Hasbach for their technical assistance in preparing the MPs, Gabi Grabs for her immunohistochemical contribution, and Robert Ivkic for his technical assistance in lymph node imaging.


   FOOTNOTES
 
See also Science to Practice in this issue.

Abbreviations: FITC = fluorescein isothiocyanate, IgM = immunoglobulin M, MP = microparticle, SAE = stimulated acoustic emission

Author contributions: Guarantors of integrity of entire study, P.H., M.R.; study concepts and design, all authors; literature research, P.H., N.D.; experimental studies, all authors; data acquisition and analysis/interpretation, P.H., M.R., N.D.; statistical analysis, P.H., M.R.; manuscript preparation and definition of intellectual content, P.H.; manuscript editing, P.H., M.R., M.S.; manuscript revision/review and final version approval, all authors


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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