Preparation of ^99mTc-Labeled Mannan-S-Cysteine and Effect of Molecular Size of Mannan on Its Biodistribution

Abstract

Macrophage mannose receptor (MMR/CD206) is a promising target for the detection and identification of sentinel lymph node (SLN). MMR-targeting probes have been developed using mannosylated dextran, however, impairment of efficient targeting of SLN was often caused because of retention of injection site in which macrophages and dendritic cells exist. In this study, we prepared new MMR-targeting probes from yeast mannan (85 kDa), and its bioditribution was investigated. In-vivo evaluation showed that 11.9% of injected dose of ^99mTc-labeled mannan-S-cysteines (^99mTc-MSCs) was accumulated in popliteal lymph node (the SLN in this model), however, significant level of radioactivity (approximately 80%) was remained in injection site. Interestingly, ^99mTc-labeled low molecular weight mannan-S-cysteine mannan (^99mTc-LSC) prepared from 50 and 25 kDa mannan showed a decreased specific accumulation of ^99mTc-LSC in the popliteal lymph node, while the radioactivity at the injection site remained unchanged. These results suggest that the molecular size, or nature/shape of the sugar chain is important for the specific accumulation of ^99mTc-MSC in popliteal lymph node.

INTRODUCTION

The sentinel lymph node (SLN) is defined as the first and closest lymph node that receives lymphatic flow (as well as metastatic tumor cells) from primary tumor sites.¹⁾ To identify these SLN prior to biopsy, single photon emission computed tomography with X-ray computed tomography (SPECT/CT) is employed for detection and identification of the SLN and is routinely used to improve diagnosis and staging.^2–5) The macrophage mannose receptor (MMR) is highly expressed on the surface of the macrophage cells abundantly resident in the lymph nodes.⁶⁾ MMR has been recognized as multispecific lectin that can recognize not only monosaccharide including glucose, mannose (Man), fucose, N-acetylglucosamine (GlcNAc) but also polysaccharides such as mannan and chondroitin sulfate.^7–9) Therefore, ^99mTc-labeled mannosylated-dextran’s have been developed because the specific binding of MMR-targeting probes to macrophage cells contribute to its high and persistent uptake in the SLN.^10–13) These compounds consist of a dextran backbone to which several mannose units are added, for recognition by the MMR with high affinity, and chelating agents are incorporated for coordinating and radiolabeling with ^99mTc to produce the imaging probe. For example, ^99mTc(CO)₃-DCM20 probe comprises an dextran backbone that has S-derivatized cysteine as a chelator for labeling with the [^99mTc(OH₂)₃(CO)₃]⁺ precursor and mannose for targeting the MMR.¹⁴⁾ However, undesirable radioactivity retention of ^99mTc (CO)₃-DCM20 at the injection site was observed in vivo biodistribution study.^14,15) Considering the decreased accumulation of ^99mTc(CO)₃-DCM20 at the injection site in the presence of DCM20, development of co-injection of competitive inhibitor could be a rational strategy to reduce the undesirable radioactivity retention.¹⁵⁾

Mannan (N-linked) has been known as a natural polysaccharide linked to asparagine of cell wall proteins in yeast. Unlike dextran, mannan consists of conserved core structure (Man₈GlcNAc₂) and more diverse outer chains.^16–18) In case of Saccharomyces cerevisiae, outer chain is composed of α(1-6)-mannose backbone to which are attached side chains consisting of α(1-2)-, α(1-3)-, α(1-2 and -6) linked mannose and few phosphate group. Considering the structural difference between dextran and mannan, ^99mTc-radiolabelled mannan derivative may exhibit a different behavior in-vivo depending on the particle size and glyosidic linkage. It is also important to investigate the effect of varying the molecular size of mannan on its SLN localization characteristics in order to develop a more effective biologically targeted SLN imaging agent.

Herein we describe the synthesis and characterization of a ^99mTc-labeled mannan-S-cysteine, as well as the effect of varying the molecular weight of mannan used on the apparent affinity for MMR. Additionally, the in-vivo behavior of these ^99mTc-labeled mannan-S-cysteine derivatives in identifying the SLN in an in-vivo mouse model via SPECT/CT imaging is described.

MATERIALS AND METHODS

Materials

Recombinant human macrophage mannose-receptor was purchased from R&D Systems, Inc. (Minneapolis, MN, U.S.A.). Mannan from Saccharomyces cerevisiae was obtained from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). Shodex STANDARD P-82 containing pullulan polysaccharides of molecular weight standards 200, 107, 47.1 and 21.1 kDa were obtained from Showa Denko Co. (Tokyo, Japan) and TiO₂ (anatase type, average particle size, 50 µm) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). ^99mTc-pertechnetate (^99mTcO₄⁻) was obtained as an eluted in saline solution from a ⁹⁹Mo-^99mTc generator available from FUJIFILM RI Pharma Co., Ltd. (Chiba, Japan). All other reagents used in the experiments were purchased from Nacalai Tesque, Inc. (Kyoto, Japan), Wako Pure Chemical Industries, Ltd., or Sigma-Aldrich Co., and were used without further purification.

Determination of Molecular Size and Particle Size of Polysaccharides

The average molecular weight of the polysaccharides was estimated by HP-SEC using a HPLC system consisting of a Hitachi L-6000 pumps, equipped with a Shimamura Tech YRD-880 refractive index detector. The chromatography was performed using an Asahipak 510 HQ column (7.6 mm, i.d. × 300 mm) (Showa Denko Co.) that was eluted with 10 mM ammonium bicarbonate at a flow rate of 0.3 mL/min. Calibration curves for molecular weight determinations were performed using Shodex STANDARD P-82 standards. The particle sizes of the polysaccharides were determined by dynamic light scattering (DLS) method on the MICROTRAC 9340 UPA-UT151 (Nikkiso Co., Ltd., Tokyo, Japan) equipped with a semi-conductor laser at the wavelength of 780 nm.

Preparation of Low Molecular Weight Mannan

Photochemical reactions were performed as described previously^19,20) using a photochemical reaction apparatus (Sen light corporation, Osaka, Japan) consisting of a VG 1500 reaction tank with 5 inlets, a light source (high pressure mercury lamp HL 100 CH-4, 100 W), a power source (HB100P-1) and Pyrex glass JW-1G. Briefly, 20 mg of mannan was dissolved in 2 mL distilled water with 2 mg of titanium dioxide (TiO₂) particles in a Pyrex glass tube. The sample tube was placed in the photochemical reaction chamber and exposed to light within 2 cm distance from the lamp unit at room temperature. After the specified time, the sample was centrifuged at 1800 × g for 5 min at 20°C and the supernatant was collected and filtered through Millex^®-LH 0.45 µm filter (Millipore Co., Billerica, MA, U.S.A.) to eliminate any remaining TiO₂ particles. The final product solution was dialyzed and lyophilized for later analysis and use.

Mass Spectrometry of Partially Methylated Alditol Acetates Derived from Mannan

Partially methylated alditol acetates (PMAAs) from mannan were prepared according to the method of Anumula and Taylor.²¹⁾ Gas chromatography-mass spectrometric spectrometry (GC-MS) analysis was carried out using a Hewlett-Packard gas chromatograph Model 6890 series II. MS were obtained by electro impact ionization (70 eV) with the following program parameters: column, HP-5MS (0.25 µm film thickness, 0.25 mm i.d. × 30 m); carrier gas, helium at 37 cm/s; split less sample injection; column oven temperature program: 3 min at 100°C, with an increase at 4°C/min to 160°C, 1 min at 160°C followed by an increase at 0.5°C/min to 180°C, and a final increase at 20°C/min to 260°C and held at 10 min at 260°C. Peak assignment was performed using Wiley’s registry of mass spectral database.

Synthesis of Allyl Mannan

Addition of allyl group to the mannan backbone was performed following the procedure outlined by Pirmettis et al.¹⁴⁾ with minor modifications. Mannan (50 mg) was dissolved in 250 µL of distilled water, followed by the addition of 125 µL of 2.5 M NaOH, 1 mg of sodium borohydride and 15.2 µL of allyl bromide. After mixing, the solution was incubated at 50°C for 3 h while maintaining the pH at 11 by addition of 2.5 M NaOH as required. After completion of the reaction, the solution was neutralized to pH 7.0 with 2.5 M acetic acid and diluted with 500 µL of water. The resulting products were filtered by Millex^®-LH 0.45 µm filters (EMD Millipore Co., MA, U.S.A.) and desalted using Amicon^® Ultra 30 K ultrafiltration tubes (EMD Millipore Co., MA, U.S.A.) following the manufacturer’s recommendations. Finally, the purified solution was collected and lyophilized for subsequent analysis and use.

Synthesis of Mannan-S-Cysteine (MSC)

L-Cysteine derivatization of the prepared allyl mannan was performed according to the Pirmettis et al.¹⁴⁾ with minor modifications. To allyl mannan (20 mg), dissolved in 100 µL of distilled water, was added 14.3 mg of L-cysteine hydrochloride monohydrate and 1.2 mg of ammonium persulfate. The resulting solution was stirred for 4 h at 50°C under a nitrogen atmosphere. The pH was then adjusted with 0.1 N NaOH to pH 4.0, and the reaction mixture was held at room temperature for an additional 24 h. Following this incubation, the volume was adjusted to 500 µL with 0.02 M sodium acetate pH 4.0 and filtered using a Millex^®-LH 0.45 µm filter (EMD Millipore Co.). After filtration, the buffer was first exchange with 0.02 M sodium acetate (pH 4.0) using Amicon^® Ultra 30 K Devices (EMD Millipore Co., MA, U.S.A.) followed by buffer exchanges with 2.5 mL (5 vol.) of 0.02 M sodium acetate (pH 4.0), 2.5 mL (5 vol) of 0.1 M bicarbonate (pH 9.0) and 2.5 mL (5 vol) of distilled water, respectively. Subsequently the washed and purified solution was collected and lyophilized.

Preparation of Rhenium-MSC

The tetraethylammonium [tribromotricarbonylrhenate] salt, ([Et₄N]₂[Re(CO)₃Br₃]), was prepared from [Re(CO)₅Br] as described by Pirmettis et al.¹⁴⁾ [Et₄N]₂[Re(CO)₃Br₃] (0.38 mg) was added to a solution of MSC (4 mg) dissolved in 2 mL of water. This sample mixture was incubated at 50°C for 16 h followed by filtration through a Millex^®-LH 0.45 µm filter (EMD Millipore Co.), and desalted using Amicon^® Ultra 30 K centrifugal filtration device (EMD Millipore Co.). The samples were further washed 10 times using 500 µL of distilled water. The purified solution was then collected and lyophilized for latter analysis and use.

Structural Analysis by ¹H-NMR Spectroscopy

Each sample was kept in a desiccator over phosphorus pentoxide in vacuo overnight at room temperature prior to use. The thoroughly dried samples were dissolved in 0.5 mL of D₂O (99.96%), centrifuged at 2000 × g for 15 min and transferred to an NMR tubes (5.0 mm o.d. × 25 cm; Wilmad Glass Co., Buena, NJ, U.S.A.). ¹H-NMR spectra were recorded at 60°C on a JNM-400 A spectrometer equipped with a 5-mm field-gradient tunable probe with standard JEOL software. The HOD signal was suppressed by pre-saturation for 1.5 s.

Preparation of ^99mTc-MSC and Its Stability

The precursor [^99mTc(OH₂)₃(CO)₃]⁺ complex used for ^99mTc-labeling the S-cysteine derivatized mannans was prepared as described previously.²²⁾ A 2 × 10⁻⁶ M solution of MSC in 0.1 M acetate (30–50 µL) was mixed with equivalent volume containing purified [^99mTc(OH₂)₃(CO)₃]⁺ solution (1.27–2.11 MBq) and incubated at 80°C for 60 min. Post ^99mTc-labeling the reaction mixture containing ^99mTc(CO)₃-MSC was purified from the unreacted [^99mTc(OH₂)₃(CO)₃]⁺ complex by Sephadex™ G-50 spin column. The radioactivity of the ^99mTc-labeled MSC was determined by TLC with Silica Gel 60 F254 TLC (Aluminium Sheets (20 ×20 cm); EMD Millipore Co.) with methyl ethyl ketone (MEK) as the developing buffer. Rf values of unreacted [^99mTc(OH₂)₃(CO)₃]⁺ and ^99mTc-MSC are 1 and 0, respectively.

Binding Study of Polysaccharides to MMR

Kinetic binding studies between polysaccharides and MMR were performed using an Octet RED 96 biolayer interferometry instrument (Primetech Corporation Ltd., Tokyo, Japan). Because mannan has asparagine residue at the reducing end,¹⁷⁾ mannan and its derivatives were immobilized to amine reactive AR2G tips of the interferometer system using standard N-[3-(Dimethylamino)propyl]-N′-ethylcarbodiimide (EDC)/N-hydroxysuccinimide (NHS)-mediated chemistry. The amine reactive AR2G tips were hydrated with running buffer containing 1 mM CaCl₂, 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) pH 7.4, 150 mM NaCl, 0.005% surfactant P20 and were later reacted with 20 mM EDC and 10 mM NHS. After incubation for 5 min, the NHS activated AR2G tips were incubated with 1 mg/mL of the desired polysaccharides for 10 min. The reaction was terminated using a 1 M ethanolamine stop solution. The ligand-coated sensor tips were subsequently immersed in regeneration solution (500 mM NaCl) for 5 s and then immersed in the running buffer for 5 s. This washing step was repeated 3-times to obtain the desired ligand-coated sensor tips. The immobilized mannan oligosaccharides in running buffer were incubated with MMR at the specified concentrations to obtain binding sensorgrams. The association and dissociation steps of the aligned data were analyzed by curve fitting which used a 1 : 1 model.

Biodistribution Studies

All animal studies were conducted in accordance with institutional guidelines and under approved protocols from Chiba University Animal Care Committee. ^99mTc-labeled MSC (2 × 10⁻¹² mol MSC; 11.1 kBq ^99mTc) in 20 µL of 0.1 M phosphate (pH 7.0) was prepared prior to use. The in-vivo biodistribution studies in all mice were performed by a subcutaneous injection of ^99mTc-labeled MSC in the hind footpad of 6-week old ddY mice. Four groups of four mice each were administrated 11.1 kBq of ^99mTc-labeled MSC in the rear footpad followed by a mild massage of the footpad for 30 s post injection. To facilitate the visualization of the lymph nodes for dissection, at 45, 165, 345 and 1425 min post injection of the ^99mTc-labeled probe, a 20 µL aqueous solution of 2% Patent Blue dye was administrated to the same footpad. At 15 min post injection of the blue dye, the animals were sacrificed and the popliteal, inguinal and lumber lymph nodes, the footpad of the injection site and other relevant tissues and organs were harvested and weighed in preweighted test tubes. After weighing each of the organ/tissue containing tubes, the radioactivity present in each of the particular excised tissues/organs was determined using a Wizard3” 1480 Automatic Gamma Counter (PerkinElmer, Inc., MA, U.S.A.). The percentage injected dose per gram of tissue (%ID/g) present in each tissue/organ was then calculated.

SPECT/CT Imaging

Mice weighing 20 – 25g were used for SPECT imaging studies. Prior to anesthesia and imaging, each mouse received in the rear footpad, a 20 µL subcutaneous injection of a solution containing 370 kBq of ^99mTc-labeled MSC (2 × 10⁻¹² mol). At the appropriate time, the mice were anesthetized with isoflurane and SPECT/CT images were acquired at 1 and 3 h post-injection with a SPECT/CT imaging system equipped with five pinhole (0.5 mm) collimators (Triumph SPECT4/CT, TriFoil Imaging Inc., CA, U.S.A.). Acquisition time was 30 min for each mouse to obtain adequate count sensitivity. 3D Images were reconstructed from the collected data for each mouse individually using the manufactured provided software. Three mice were used for each imaging studies.

RESULTS AND DISCUSSION

Characterization of Mannan Structure

In synthesizing ^99mTc-labeled mannan for use in SLN, it is important to understand the glycosidic linkage of mannan and its molecular weight as this may affect its in-vivo behavior. GC-MS analysis of the reaction products of PMAAs derived from the polysaccharides has been recognized as a powerful tool to investigate the glycosidic linkages.²³⁾ Based on this technique, mannan was derivatized to PMAAs and the resulting products were subsequently subjected to GC-MS analysis. The resulting peaks identified were assigned by Wiley’s registry of mass spectral database (Supplementary Fig. 1). Peaks corresponding to 2,3,4,6-tetra-, 3,4,6-tri-, 2,4,6-tri-, 2,3,4-tri-, and 3,4-di-O-methyl-D-mannitol were detected, and mannan used in this study is found to be consisted of 33% of non-reducing end residues, 25% of α(1-2)-, 8.1% of α(1-3)-, 2.4% of α(1-6)-, 32% of α(1-2 and -6) glycosidic linkages (Table 1). Additionally, subjecting the mannan to GPC-HPLC (data not shown), the molecular size of the mannan was found to be 85 kDa (ca. 525 mannose residues). Based on the structural information of mannan,¹⁶⁾ we are able to estimate that mannan consisted of ca. 180 mannose residues as a α(1-6)-mannose backbone and ca. 340 mannose residues as branches. Moreover, we also estimated the particle size of mannan using dynamic light scattering and determined the average particle size of intact mannan to be 8.4 ± 0.2 nm (data not shown), which is similar to the dextran-based radiopharmaceuticals such as a DTPA-mannosylated-dextran (7.1 ± 0.9 nm), MAG₃-mannosyl-dextran (5.5 ± 2.4 nm), pyrazolyl-mannosylated-dextran (7.0 ± 0.7 nm) and mannosylated dextran DCM20 (8.3 ± 0.5 nm).^{10,12–14,24)} Among them, DCM20 was prepared from dextran-8 (MW 11.8 kDa) was used as a template. Based on the structural information of dextran derived from L. mesenteroides NRRL-B,²³⁾ it is able estimate that dextran-8 consists of ca. 57 glucose residues as a sugar backbone and ca. 16 glucose residues as branches.

Table 1. Determination of Partial Alditol Acetates from Mannan and Their Derivatives

Peak	O-Methylated alditol acetates	Peak area (%)
		Mannan	MSC (11% allylated)	MSC (57% allylated)	LMWMa (50 kDa)	LMWMa (25 kDa)
1	2,3,4,6-Tetra-O-methylmannitol	33	32	22	27	26
2	3,4,6-Tri-O-methylmannitol	25	22	18	23	24
4	2,4,6-Tri-O-methylmannitol	8.1	8.0	7.5	7.0	7.2
5	2,3,4-Tri-O-methylmannitol	2.4	3.0	7.8	3.7	1.8
12	3,4-Di-O-methylmannitol	32	35	45	40	41

Degradation of Mannan by Photochemical Reaction

We have previously reported the photochemical depolymerization of natural polysaccharides such as alginate, pectin, K5 heparosan and heparin, and that the resulting products maintained intact core structures and their biological functions.^19,20,25,26) To examine the effect of molecular size of mannan on its biodistribution, partial photolysis with visible light (>370 nm) was performed in distilled water containing titanium dioxide (TiO₂). The resulting polysaccharides at various times points were collected and molecular weight of them were determined. As shown in Figs. 1A and B, preparation of 50 and 25 kDa of mannan (low molecular weight mannan: LMWMa) required 9 and 15 h exposure, respectively. The ¹H-NMR signals for 50 or 25 kDa LMWMa showed the same chemical shift values and signal intensities as observed for the intact mannan (Fig. 1C). These results suggest that the preparation of LMWMa was successfully achieved without significant changes to the overall structure of the oligosaccharide polymer. Based on the result of GC-MS analysis techniques, we estimate that in LMWMa (25 kDa), the α(1-6)-mannose backbone consisted of ca. 66 mannose residues and ca. 88 mannose residues are present as branches. Thus, comparatively, the chain length of sugar backbone between LMWMa (25 kDa) and dextran-8 (11.8 kDa) seem similar.¹⁴⁾

Fig. 1. Preparation and Characterization of Low Molecular Weight of Mannan

(A) Molecular weights of photodegraded mannans at specified time. Photochemical depolymerization of mannan in the presence of titanium dioxide is performed as described under “Materials and Methods.” After specified time, the sample was centrifuge at 1800 × g for 5 min at 20°C and the supernatant was filtered and dialyzed. Resulting depolymerized mannan was subjected to GPC-HPLC. (B) Chromatograms of mannan and photodegraded mannans at 9 and 15 h exposure. (C) ¹H-NMR spectra of mannan, LMWMa (50 kDa) and LMWMa (25 kDa).

Synthesis of MSC and Its Rhenium Complexes

Preparation of mannan-S-cysteine (MSC) was performed as described by Pirmettis et al.¹⁴⁾ The synthesis involved the initial synthesis of allyl mannan, followed by the S-derivatization of allyl mannan with cysteine (Fig. 2A). In all steps the products were purified through ultrafiltration and their identity was established by ¹H-NMR. The ¹H-NMR of allylated mannan was examined, and three peaks resonating at 6.02, 5.37 and 4.19 ppm, corresponding to allyl group, were observed (Fig. 2B). A comparison of the ¹H-NMR allyl peak integral intensity (6.02 ppm) to the integral of the anomeric proton of both the substituted and the unsubstituted mannose units of mannan indicates that ca. 11% of the mannose units were allylated. Next, mannan-S-cysteine (MSC) was prepared and isolated. The isolated MSC displayed a distinct absence of the allyl resonances and the appearance of characteristic peaks belonging to the propylene ether chain at 2.72 and 1.93 ppm. Additionally, the βCH₂ protons of the introduced cysteine moiety post reaction, were observed in the ¹H-NMR spectrum at 2.9–3.2 ppm.

Fig. 2. Synthesis of Rhenium and Technetium-99m Complexes (A) and ¹H-NMR Spectra of Mannan, Allyl-Mannan, MSC and Re-MSC (B)

LMWMa-S-cysteine (i.e. LSC) was also prepared from LMWMa (50 kDa) and (25 kDa) according to the above-mentioned method, and resulting products were also synthesized and confirmed by ¹H-NMR spectroscopy (Supplementary Fig. 2). Composition ratio of allylated mannose unit in LMWMa are ca. 13% (50 kDa) and ca. 9% (25 kDa), respectively (Supplementary Figs. 2B, F).

Pirmettis et al. reported that ca. 41% of the glucose units were allylated in DCM20.¹⁴⁾ Thus, we also prepared a highly allylated mannan (57%) with S-derivatized cysteine, however, its affinity for MMR protein was significantly decreased (data not shown). PMAAs derived from MSC were analyzed using GC-MS analysis to determine the structure of MSC in which the mannose units were modified with allyl group. The composition of 2,3,4,6-tetra-, 3,4,6-tri-, 2,4,6-tri-O-methyl-D-mannitol derived from MSC (57% allylated) decreased, while 2,3,4-tri-, 3,4-di-O-methyl-D-mannitol increased compared with intact mannan (Table 1). These results suggest that the incorporation of the allyl groups on mannose residues took place on the outer chains.

Rhenium is the group VIIB congener of technetium and is often used as a nonradioactive surrogate for the radioactive element technetium. Thus, Re (CO)₃-MSC was prepared by addition [NEt₄]₂[fac-ReBr₃ (CO)₃] to MSC. The purified material was then subjected to ¹H-NMR (Fig. 2B). The peaks corresponding to the [Re (CO)₃]-coordinated cysteine, namely the βCH₂ protons and the coordinated OCH₂CH₂CH₂S were observed. The intensities of propylene ether chain peaks at 2.72 and 1.93 ppm were also slightly reduced. Based on the degree of decline in the of peak intensities at 2.72 ppm, we estimate that on average 5–8 fac-[Re(CO)₃]⁺ metal cores are coordinated per molecule of mannan.

Effect of Molecular Size and S-Derivatization on Binding Activity of Mannan for MMR

Quantitative kinetic analysis was performed to examine the effect of molecular size and S-derivatization of mannan on the affinity towards the MMR (Fig. 3). As a result, K_d values of intact mannan and MSC for the MMR were 4.14 ± 0.60 and 2.50 ± 0.84 nM, respectively (Fig. 3B). Thus, interaction between mannan and MMR was not significantly influenced by derivatization with S-cysteine onto the mannan backbone of MSC. In case of LSC, affinity for MMR is decreased in molecular weight dependent manner. K_d values of LSC (50 kDa) and LSC (25 kDa) were 21.9 ± 1.80 and 73.6 ± 20.4 nM, respectively. Resultant decrease of their affinity for MMR is attribute to the significant increase of k_dis values. It has been reported that binding mode of mannan to MMR was different from mannose and mannosylated bovine serum albumin (Man₂₃-BSA).⁹⁾ MMR is a type I transmembrane protein consisted of a fibronectin type II repeat and eight Ca²⁺-dependent carbohydrate-recognition domains (CRDs). While CRD 4 of MMR is critical domain to interact with mannan monosaccharide and Man₂₃-BSA, CRDs in tandem (4–8), especially CRDs 4 and 5 are required for the binding of MMR to yeast mannan.⁹⁾ It might be thought that LSCs cannot fit the geometrical arrangement of binding site in CRDs 4–8 of MMR.

Fig. 3. Sensorgrams for the Binding of MMR to Mannan

(A) Mannan, MSC and LMWMa-S-cysteine (LSC) were immobilized to amine reactive AR2G tips as described in “Materials and Methods.” Immobilized mannan and its derivatives were incubated with MMR at the specified concentrations. Sensorgrams obtained with various kinds of polysaccharides immobilized sensor tips were overlaid using a Octet software version 6.4. (B) The k_on, k_dis and k_D values were determined using a curve fitting which used 1 : 1 model as described under “Materials and Methods.” The values for each mannan and its derivatives were expressed as the mean ± standard error (S.E.) of three different experiments.

In-Vivo Biodistribution and SLN Mapping Studies in Mice Using ^99mTc-MSC, ^99mTc-LSC (50 kDa) and ^99mTc-LSC (25 kDa) with SPECT/CT Imaging

The in-vivo biodistribution study of the ^99mTc(CO)₃-MSC was performed through the determining the amount of the radiolabeled ^99mTc(CO)₃-MSC in various excised organs and tissues. As shown in Table 2, the uptake in popliteal lymph node (SLN in this case) for the ^99mTc(CO)₃-MSC is 11.9% ID/g at 1 h post injection. This accumulation of ^99mTc(CO)₃-MSC in popliteal lymph node stably remains for up to 24 h with a significantly small amount traversing to the 2nd and 3rd lymph nodes. Accumulation and stability of ^99mTc(CO)₃-MSC in popliteal lymph node is comparable to DCM20, however, the radioactivity level of ^99mTc(CO)₃-MSC at the liver (0.25% ID/g at 1 h) is much lower than that of DCM20 (1.2% ID/g).¹⁴⁾ Minor accumulation levels in stomach (0.01% ID/g), intestines (0.09%) and kidney (0.03%) are also observed. These results indicate that the ^99mTc(CO)₃-MSC is stable in vivo and no appreciable amount is released and washed out into the systemic circulation. In-vivo SPECT/CT imaging experiments in intact mice were also conducted after a subcutaneous injection in the footpad of ^99mTc(CO)₃-MSC. The 3D SPECT/CT images from these studies at 1 and 3 h post injection are illustrated (Fig. 4). The popliteal lymph node is clearly delineated with intense uptake (Fig. 4A). Clear but relatively less accumulation of ^99mTc(CO)₃-MSC at the 2nd and 3rd LN are also observed. Considering the importance of particle size of other radiopharmaceuticals used in SLN identification and mapping,²⁴⁾ we also evaluated the ^99mTc(CO)₃-LSC (50 kDa) and ^99mTc(CO)₃-LSC (25 kDa) in in-vivo biodistribution and SPECT/CT imaging studies (Table 2). Both the ^99mTc(CO)₃-LSC (50 kDa) and ^99mTc(CO)₃-LSC (25 kDa) showed significantly less uptake and localization in the popliteal lymph node. This result is consistent with the data that affinities of LSC (50 kDa) and LSC (25 kDa) for MMR are lower than that of mannan and MSC (Fig. 3). In addition, increased accumulation of ^99mTc(CO)₃-LSC in the liver and kidney are also observed. It should be noted that accumulation level of ^99mTc(CO)₃-LSC (50 kDa) (1.91%ID/g at 1 h) and ^99mTc(CO)₃-LSC (25 kDa) (1.12%ID/g at 1 h) at the liver are comparable to the accumulation level of DCM20 whose molecular weight is 22 kDa.^{14) 99m}Tc(CO)₃-LSC (50 kDa) and ^99mTc(CO)₃-LSC (25 kDa) also show early accumulate in the bladder at 1 h indicating that ^99mTc(CO)₃-LSC (50 kDa) and ^99mTc(CO)₃-LSC (25 kDa) are being cleared from the lymphatic’s and slowly excreted into the urine (Fig. 4B, C). In contrast to this, there is little change in the accumulation of these ^99mTc(CO)₃-LSC in the blood and injection site, where they show low activity and increased retention respectively and similar to that observed with higher molecular weight ^99mTc(CO)₃-MSC. These results strongly suggest that the molecular weight of mannan is an important factor in the specific accumulation and clearance of ^99mTc-labelled mannan in the popliteal lymph node.

Table 2. Biodistribution of Radioactivity in Mice after Injection of ^99mTc(CO)₃-MSC, ^99mTc(CO)₃-LSC (50 kDa) and ^99mTc(CO)₃-LSC (25 kDa)

		Time after injection (h)
		1	3	6	24
Popliteal lymph node	MSC	11.9 ± 5.10	11.1 ± 2.55	10.3 ± 4.44	13.0 ± 3.46
	LSC (50 kDa)	6.47 ± 1.99	4.71 ± 1.28	4.92 ± 1.01	6.23 ± 0.92
	LSC (25 kDa)	3.78 ± 2.73	4.81 ± 1.22	4.48 ± 0.49	2.59 ± 1.14
2nd LN	MSC	1.70 ± 2.12	0.45 ± 0.29	0.78 ± 0.85	0.94 ± 0.83
	LSC (50 kDa)	1.08 ± 0.21	0.88 ± 0.28	1.17 ± 0.34	0.78 ± 0.48
	LSC (25 kDa)	0.42 ± 0.28	0.83 ± 0.43	0.58 ± 0.18	0.31 ± 0.23
3rd LN	MSC	0.91 ± 0.97	0.35 ± 0.45	0.91 ± 0.83	0.88 ± 0.86
	LSC (50 kDa)	0.97 ± 0.17	1.31 ± 0.58	1.14 ± 0.77	0.51 ± 0.18
	LSC (25 kDa)	0.64 ± 0.52	0.57 ± 0.11	0.45 ± 0.15	0.32 ± 0.30
Blood	MSC	0.06 ± 0.06	0.02 ± 0.01	0.05 ± 0.02	0.04 ± 0.01
	LSC (50 kDa)	0.07 ± 0.01	0.05 ± 0.01	0.04 ± 0.00	0.04 ± 0.02
	LSC (25 kDa)	0.05 ± 0.00	0.04 ± 0.00	0.05 ± 0.01	0.06 ± 0.02
Liver	MSC	0.25 ± 0.18	0.20 ± 0.03	0.46 ± 0.18	0.47 ± 0.14
	LSC (50 kDa)	1.91 ± 0.24	1.84 ± 0.32	2.00 ± 0.26	1.25 ± 0.10
	LSC (25 kDa)	1.12 ± 0.28	1.14 ± 0.05	1.52 ± 0.15	1.84 ± 0.56
Spleen	MSC	0.32 ± 0.35	0.20 ± 0.03	0.47 ± 0.20	0.33 ± 0.10
	LSC (50 kDa)	1.03 ± 0.12	1.00 ± 0.22	0.98 ± 0.41	0.04 ± 0.01
	LSC (25 kDa)	0.76 ± 0.54	0.80 ± 0.60	0.90 ± 0.69	0.81 ± 0.47
Kidney	MSC	0.03 ± 0.02	0.03 ± 0.03	0.10 ± 0.11	0.03 ± 0.01
	LSC (50 kDa)	1.26 ± 0.23	1.12 ± 0.17	1.16 ± 0.22	0.09 ± 0.05
	LSC (25 kDa)	1.29 ± 0.26	1.29 ± 0.20	1.44 ± 0.27	1.16 ± 0.59
Pancreas	MSC	0.38 ± 0.36	0.33 ± 0.12	0.60 ± 0.38	0.73 ± 0.30
	LSC (50 kDa)	0.58 ± 0.12	0.70 ± 0.39	0.51 ± 0.07	0.02 ± 0.01
	LSC (25 kDa)	0.55 ± 0.49	0.27 ± 0.06	0.80 ± 0.57	0.28 ± 0.06
Heart	MSC	0.03 ± 0.03	0.01 ± 0.00	0.02 ± 0.01	0.01 ± 0.01
	LSC (50 kDa)	0.33 ± 0.12	0.75 ± 0.92	0.33 ± 0.14	0.00 ± 0.00
	LSC (25 kDa)	0.21 ± 0.02	0.17 ± 0.02	0.20 ± 0.01	0.18 ± 0.05
Lung	MSC	0.04 ± 0.05	0.02 ± 0.01	0.04 ± 0.03	0.03 ± 0.01
	LSC (50 kDa)	0.25 ± 0.07	0.54 ± 0.40	0.22 ± 0.07	0.00 ± 0.00
	LSC (25 kDa)	0.10 ± 0.01	0.08 ± 0.02	0.10 ± 0.03	0.08 ± 0.02
Stomach	MSC	0.01 ± 0.02	0.01 ± 0.00	0.02 ± 0.01	0.05 ± 0.04
	LSC (50 kDa)	0.49 ± 0.15	0.40 ± 0.04	0.62 ± 0.12	0.03 ± 0.01
	LSC (25 kDa)	0.18 ± 0.01	0.15 ± 0.01	0.32 ± 0.08	0.59 ± 0.76
Intestine	MSC	0.09 ± 0.08	0.07 ± 0.01	0.20 ± 0.12	0.28 ± 0.15
	LSC (50 kDa)	0.32 ± 0.11	0.41 ± 0.11	0.66 ± 0.11	0.08 ± 0.00
	LSC (25 kDa)	0.12 ± 0.03	0.12 ± 0.02	0.26 ± 0.03	0.81 ± 0.88
Injection site	MSC	84.3 ± 12.4	74.1 ± 9.11	81.6 ± 15.5	74.4 ± 5.62
	LSC (50 kDa)	72.8 ± 6.14	65.5 ± 10.3	62.5 ± 3.18	62.8 ± 7.54
	LSC (25 kDa)	82.0 ± 4.62	81.3 ± 2.07	77.3 ± 6.98	69.4 ± 5.93

Tissue radioactivity is expressed as % ID/g for each group (n = 4–5); results are expressed as the mean ± standard deviation (S.D.).

Fig. 4. SPECT/CT Images in Mice after Subcutaneous Injection of ^99mTc(CO)₃-MSC, ^99mTc(CO)₃-LSC (50 kDa) and ^99mTc(CO)₃-LSC (25 kDa)

After 1 or 3 h post-injection of each ^99mTc-labeled compound, mice were anesthetized using isofluran. SPECT imaging and X-ray CT imaging were performed by use of SPECT/CT system equipped with a five pinhole (0.5 mm) collimator, and 1 h or 3 h data acquisition was performed. IS, injection site; 2nd, secondary (inguinal) lymph node; 3rd, third (lumber) lymph node; BL, bladder.

It has been known that mannan is recognized by many pattern recognition receptors (PPRs) that bind pathogen-associated molecular patterns (PAMPs) including fungal cell wall components. For example, MMR binds terminal mannose,^7,9) dectin-2 recognizes the core structure (Man₉GlcNAc₂),²⁷⁾ DC-SIGN binds α(1-2)- and α(1-3)-mannosides.²⁸⁾ MMR, dectin-2 and DC-SIGN are expressed macrophages as well as dendritic cells which are localized in popliteal lymph node and injection sites.^15,29) Thus, LMWMa (50 kDa) and LMWMa (25 kDa) may be useful for the material to develop the competitive inhibitor to reduce the undesirable radioactivity retention of MSC and DCM20. Experiments are now in progress to examine the development of co-injection of LMWMa (50 kDa) or LMWMa (25 kDa) as a competitive inhibitor to reduce the undesirable radioactivity retention of MSC and DCM20 at the injection site.

Acknowledgments

The authors would like to thank to Drs. Kenjirou Higashi and Kunikazu Moribe (Chiba University) for measurement of particle size of mannan by dynamic light scattering. The authors are grateful for support by Special Funds for Education and Research (Development of SPECT Probes for Pharmaceutical Innovation) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest

The authos declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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