Comparative Study of a Series of 99mTc(CO)3 Mannosylated Dextran Derivatives for Sentinel Lymph Node Detection

Sentinel lymph node detection (SLND) is rapidly entering common practice in the management of patients with tumors. The introduction of mannose molecules to 99mTc-labeled dextrans, so far, showed that the sentinel node could trap these agents due to their recognition by the mannose receptors of lymph node macrophages. The current study aimed to synthesize, characterize, and biologically evaluate a series of mannosylated dextran derivatives labeled with 99mTc for potential use in SLND. The compounds were designed to have a dextran with a molecular weight of 10–500 kDa as a backbone, S-derivatized cysteines, efficient SNO chelators, and mannose moieties for binding to mannose receptors. They were successfully synthesized, thoroughly characterized using NMR techniques, and labeled with the fac-[99mTc(CO)3]+ synthon. Labeling with high yields and radiochemical purities was achieved with all derivatives. In vivo biodistribution and imaging studies demonstrated high uptake in the first lymph node and low uptakes in the following node and confirmed the ability to visualize the SLN. Among the compounds studied, 99mTc-D75CM demonstrated the most attractive biological features, and in combination with the high radiochemical yield and stability of the compound, its further evaluation as a new radiopharmaceutical for sentinel lymph node detection was justified.


Introduction
Over the last decade, research on developing new radiopharmaceuticals has focused on the selective binding of a radiolabeled biomolecule to a receptor. Specifically, the biomolecule acts as a vector that transports the radionuclide to the tissue overexpressing the receptor to enable imaging and diagnosis with a SPECT or PET camera or with radiotherapy [1]. Among the SPECT isotopes currently in use, technetium-99m ( 99m Tc) remains the radionuclide of choice, having excellent physicochemical properties, easy access through generators, and low cost compared with radionuclides, the production of which requires a cyclotron ( 18 F, 123 I, 111 In, 11 C, 13 N, 15 O) [2].
The development of diagnostic radiopharmaceuticals for the precise localization of the sentinel lymph node (SLN), the hypothetical first lymph node to receive lymph and metastatic cells from the primary site of the tumor, is actively explored, aiming at identifying affected tissues that require surgical removal. The metastatic pathway of some cancers usually follows a course that begins from the nearby lymph nodes. Therefore, the dextran glucose units were allylated. Addition of cysteine to allyl dextran resulted in the quantitative formation of the dextran-cysteine derivatives (D10C-D500C). Subsequently, D10C-D500C reacted with the bifunctional reagent 2-imino-2-methoxethyl-1-thioβ-D-mannopyranoside to yield the mannosylated dextran derivatives (D10CM-D500CM). Comparison of the intensity of the anomeric protons of mannose to that of the anomeric protons of dextran showed that 53-82% of the cysteines were mannosylated. The number of cysteines and mannose units present in each DCM derivative based on NMR data are presented in Table 1. The 1 H NMR spectra of D75A, D75C, and D75CM are given in Supplementary Materials ( Figure S1) as representative of the spectra obtained with the different MW dextrans, together with a stack plot of the 1 HNMR spectra of the rest of the mannosylated dextrans ( Figure S2).
Molecules 2021, 26,4797 3 of 16 reaction of allyl bromide with dextrans D10-D500 (MW 10-500 kDa) yielded the intermediate allyl dextrans D10A-D500A, Scheme 1. Comparing the intensity of allyl peaks in the NMR spectra with those of the anomeric protons of dextran indicated that 21-35% of the dextran glucose units were allylated. Addition of cysteine to allyl dextran resulted in the quantitative formation of the dextran-cysteine derivatives (D10C-D500C). Subsequently, D10C-D500C reacted with the bifunctional reagent 2-imino-2-methoxethyl-1thio-β-D-mannopyranoside to yield the mannosylated dextran derivatives (D10CM-D500CM). Comparison of the intensity of the anomeric protons of mannose to that of the anomeric protons of dextran showed that 53-82% of the cysteines were mannosylated. The number of cysteines and mannose units present in each DCM derivative based on NMR data are presented in Table 1. The 1 H NMR spectra of D75A, D75C, and D75CM are given in Supplementary Materials ( Figure S1) as representative of the spectra obtained with the different MW dextrans, together with a stack plot of the 1 HNMR spectra of the rest of the mannosylated dextrans ( Figure S2).

Radiolabeling
Radiolabeling was effected through the employment of the 99m Tc(CO) 3 tricarbonyl core. In-house production of the fac-[ 99m Tc(CO) 3 (H 2 O) 3 ] + precursor was performed by direct addition of 99m TcO 4 − in a closed vial in the presence of CO gas and NaBH 4 as a reducing agent and by heating for 30 min at 95 • C, followed by adjustment of the pH to 7. The quality control of the radiolabeling by HPLC showed more than 97% formation of the precursor.

Radiolabeling
Radiolabeling was effected through the employment of the 99m Tc(CO)3 tricarbonyl core. In-house production of the fac-[ 99m Tc(CO)3(H2O)3] + precursor was performed by direct addition of 99m TcO4 -in a closed vial in the presence of CO gas and NaBH4 as a reducing agent and by heating for 30 min at 95 °C, followed by adjustment of the pH to 7. The quality control of the radiolabeling by HPLC showed more than 97% formation of the precursor.
Stability studies showed that the 99m Tc-complexes remained stable in their reaction mixture for up to 24 h. Incubation of 99m Tc-complexes at 37 °C with a significant excess of the amino acids cysteine or histidine that display high affinity for the fac-[ 99m Tc(CO)3] + synthon showed that more than 95% of the complexes remained intact ( Figure 2).  Stability studies showed that the 99m Tc-complexes remained stable in their reaction mixture for up to 24 h. Incubation of 99m Tc-complexes at 37 • C with a significant excess of the amino acids cysteine or histidine that display high affinity for the fac-[ 99m Tc(CO) 3 ] + synthon showed that more than 95% of the complexes remained intact ( Figure 2).
At 15 min post injection, 25% to 37% of the injected dose (ID) cleared from the injection site (footpad). At the same time, a significant percentage of the injected activity (1.61% to 4.71% ID) localized in the popliteal lymph node, the sentinel lymph node in this study. The highest popliteal lymph node uptake was measured for the 99m Tc-D10CM and 99m Tc-D75CM derivatives, 4.71 ± 0.13% and 4.20 ± 0.84% ID, respectively, while the lowest was measured for the 99m Tc-D40CM derivative (1.61 ± 0.39% ID).
Slower injection site clearance was observed from 15 to 60 min post injection. In general, after 60 min, injection site uptake remained quite stable in a range from 46% to 70% ID. On the contrary, a significant increase in the popliteal lymph node uptake at 60 min p.i. was observed, ranging between 5.80% and 15.00% ID, that remained almost constant up to 180 min p.i. (6.44 to 13.53% ID). The highest values, 15.0 ± 1.5% and 13.53 ± 0.45% ID, were measured for compound 99m Tc-D75CM at 60 and 180 min p.i., respectively ( Table 2).
The uptake of complexes in the second lymph node was in the range 0.49-1.84% ID at 15 min p.i., slowly increasing to 1.81-2.37% ID at 60 min p.i. and then remaining practically stable up to 180 min p.i. (1.49-2.30% ID). The ratio between the first and second lymph nodes was high at all time points and above 3 for most complexes. The highest ratios (8.3 and 9.0) were found for complex 99m Tc-D75CM at 60 and 180 min p.i., respectively.
At 15 min post injection, 25% to 37% of the injected dose (ID) cleared from the injection site (footpad). At the same time, a significant percentage of the injected activity (1.61% to 4.71% ID) localized in the popliteal lymph node, the sentinel lymph node in this study. The highest popliteal lymph node uptake was measured for the 99m Tc-D10CM and 99m Tc-D75CM derivatives, 4.71 ± 0.13% and 4.20 ± 0.84% ID, respectively, while the lowest was measured for the 99m Tc-D40CM derivative (1.61 ± 0.39% ID).
Slower injection site clearance was observed from 15 to 60 min post injection. In general, after 60 min, injection site uptake remained quite stable in a range from 46% to 70% ID. On the contrary, a significant increase in the popliteal lymph node uptake at 60 min p.i. was observed, ranging between 5.80% and 15.00% ID, that remained almost constant up to 180 min p.i. (6.44 to 13.53% ID). The highest values, 15.0 ± 1.5% and 13.53 ± 0.45% ID, were measured for compound 99m Tc-D75CM at 60 and 180 min p.i., respectively ( Table 2).
The uptake of complexes in the second lymph node was in the range 0.49-1.84% ID at 15 min p.i., slowly increasing to 1.81-2.37% ID at 60 min p.i. and then remaining practically stable up to 180 min p.i. (1.49-2.30% ID). The ratio between the first and second lymph nodes was high at all time points and above 3 for most complexes. The highest ratios (8.3 and 9.0) were found for complex 99m Tc-D75CM at 60 and 180 min p.i., respectively. The percentage of the radioactivity in the blood at 15 min p.i. was low (0.52-2.06% ID/g) and dropped further at 180 min p.i. (0.08-1.17% ID/g). A low concentration of radioactivity was measured in most of the organs. Specifically, the radiation in the stomach and spleen at all time points was minimal, indicating the in vivo stability of the complexes with respect to reoxidation to 99m TcO4 -and 99m TcO2. A significant amount of radioactivity was measured only in the liver, ranging from 1.08% to 3.72% ID/g at 15 min  The results are expressed as % ID/g. Mean values are reported ± SD; n = 3. * Mean % ID values are reported ± SD; n = 3.
The percentage of the radioactivity in the blood at 15 min p.i. was low (0.52-2.06% ID/g) and dropped further at 180 min p.i. (0.08-1.17% ID/g). A low concentration of radioactivity was measured in most of the organs. Specifically, the radiation in the stomach and spleen at all time points was minimal, indicating the in vivo stability of the complexes with respect to reoxidation to 99m TcO 4 − and 99m TcO 2 . A significant amount of radioactivity was measured only in the liver, ranging from 1.08% to 3.72% ID/g at 15 min p.i. to 1.79% to 4.14% ID/g at 180 min p.i. The uptake in intestines was below 1.0% ID/g for all complexes at all times studied except for complex 99m Tc-D40CM (1.68% ID/g at 180 min p.i.), indicating minimal hepatobiliary excretion. Urinary excretion was also low, as indicated by the small percentage of radioactivity in urine (1.32-5.58% ID at 180 min p.i.).
The biodistribution of the non-mannosylated compound 99m Tc-D75C was also studied. The results, presented in Table 2, show superior injection site clearance with only 24.1% ID remaining in the injection site at 180 min p.i. However, only marginal uptake was observed in the popliteal lymph node (1.04-2.43% ID) and in the second lymph node (0.53-1.72% ID). In addition, high radioactivity levels were measured in the blood at all times studied (6.16-10.13% ID/g), accompanied by significant urinary excretion (22.46 ± 3.55% ID) at 180 min p.i.
The in vivo imaging study of the 99m Tc-labeled mannosylated dextran derivative 99m Tc-D75CM, which showed the best overall biodistribution profile, as well as of the non-mannosylated 99m Tc-D75C analogue, was performed by injecting the compounds in the rear footpad. The images (Figures 4 and 5) are in agreement with the biodistribution data. Minutes after injecting 99m Tc-D75CM, the lymph nodes were clearly delineated, and the localization persisted up to 24 h after injection. The uptake in a second and a third lymph node may be due to the saturation of the mannose receptors in the small popliteal lymph node of the mouse (1-2 mg) by the excess (2 µg, 1.8 × 10 −11 moles) of non-radiolabeled D75CM injected in the imaging studies. On the contrary, after injecting the non-mannosylated compound 99m Tc-D75C, only marginal lymph node uptake was observed, followed by extensive urinary excretion. After intravenous injection of the mannosylated compound 99m Tc-D75CM ( Figure 6), a significant percentage of radioactivity was rapidly excreted in urine, but a high and persistent localization in the liver was observed. This finding is consistent with reports in the literature, referring to the mannose receptor (CD206) as being predominantly expressed by liver endothelial cells [25].

Discussion
Sentinel lymph node detection (SLND) is rapidly entering common practice in the management of patients with tumors. SLND in nuclear medicine is performed by injecting small radiolabeled particles in the area where a tumor is located. 99m Tc-labeled dextrans carrying mannoses are known to be trapped in the SLN due to their recognition by the mannose receptors of lymph node macrophages. These nanocompounds consist of a 10 kDa dextran backbone, several mannose units for recognition by the mannose receptors, and a chelating agent such as DTPA in Lymphoseek, MAG3, or pyrazolyl-diamine for coordinating 99m Tc [23,24].
In previous studies [23,24], we presented the development of a novel mannosylated dextran derivative DCM20 that can be labeled with 99m Tc using the fac-[ 99m Tc(OH2)3(CO)3] + precursor. The compound has a 10 kDa dextran as a backbone, 6 Sderivatized cysteines as efficient SNO chelators, and 24 mannose moieties for binding to mannose receptors. The biological evaluation of the 99m Tc-labelled 99m Tc(CO)3-DCM20 showed specific uptake in the mouse RAW 264.7 mannose receptor-bearing macrophage cells, rapid and high accumulation in the popliteal lymph node that remained almost stable up to 6 h, and fast clearance from the injection site.
In this work, we describe the synthesis, labeling with 99m Tc, and biological evaluation of a series of analogous mannosylated dextran derivatives ( 99m Tc-D10CM-99m Tc-D500CM) having a dextran backbone with a molecular weight of 10-500 kDa. It should be noted that the derivative 99m Tc-D10CM is essentially the same as the previously studied [23,24] compound 99m Tc(CO)3-DCM20 and is included for comparison purposes. The synthesis and characterization of the compounds D10CM-D500CM was straightforward, and no significant differences either in the percentage of allylation or in the percentage of mannosylation were noted between derivatives. Labeling with high yield and radiochemical purity was achieved with all derivatives. The stability studies of the generated 99m Tc-complexes showed that more than 95% of the original complexes were present after 6 h incubation with excess cysteine or histidine at 37 °C, demonstrating the strong li-

Discussion
Sentinel lymph node detection (SLND) is rapidly entering common practice in the management of patients with tumors. SLND in nuclear medicine is performed by injecting small radiolabeled particles in the area where a tumor is located. 99m Tc-labeled dextrans carrying mannoses are known to be trapped in the SLN due to their recognition by the mannose receptors of lymph node macrophages. These nanocompounds consist of a 10 kDa dextran backbone, several mannose units for recognition by the mannose receptors, and a chelating agent such as DTPA in Lymphoseek, MAG 3 , or pyrazolyl-diamine for coordinating 99m Tc [23,24].
In previous studies [23,24], we presented the development of a novel mannosylated dextran derivative DCM20 that can be labeled with 99m Tc using the fac-[ 99m Tc(OH 2 ) 3 (CO) 3 ] + precursor. The compound has a 10 kDa dextran as a backbone, 6 S-derivatized cysteines as efficient SNO chelators, and 24 mannose moieties for binding to mannose receptors. The biological evaluation of the 99m Tc-labelled 99m Tc(CO) 3 -DCM20 showed specific uptake in the mouse RAW 264.7 mannose receptor-bearing macrophage cells, rapid and high accumulation in the popliteal lymph node that remained almost stable up to 6 h, and fast clearance from the injection site.
In this work, we describe the synthesis, labeling with 99m Tc, and biological evaluation of a series of analogous mannosylated dextran derivatives ( 99m Tc-D10CM-99m Tc-D500CM) having a dextran backbone with a molecular weight of 10-500 kDa. It should be noted that the derivative 99m Tc-D10CM is essentially the same as the previously studied [23,24] compound 99m Tc(CO) 3 -DCM20 and is included for comparison purposes. The synthesis and characterization of the compounds D10CM-D500CM was straightforward, and no significant differences either in the percentage of allylation or in the percentage of mannosylation were noted between derivatives. Labeling with high yield and radiochemical purity was achieved with all derivatives. The stability studies of the generated 99m Tc-complexes showed that more than 95% of the original complexes were present after 6 h incubation with excess cysteine or histidine at 37 • C, demonstrating the strong ligation of the 99m Tc(CO) 3 core to the SNO cysteine ligand and the suitability of the radiotracers for biological and imaging applications.
In vivo biodistribution studies in mice demonstrated fast injection site clearance, high uptake in the first lymph node, and relatively low uptake in the following node for all mannosylated compounds. Regarding the injection site clearance, no significant improvement was observed in comparison with the previously reported compound 99m Tc(CO) 3 -DCM20. However, the uptake of 99m Tc-D75CM in the popliteal lymph node (13-15% ID) was almost 2 times higher ( Figure 3, Table S1). The specific uptake of 99m Tc-D75CM in the lymph node was supported by the low uptake of the non-mannosylated analogue 99m Tc-D75C.
In vivo imaging studies in mice with 99m Tc-D75CM were in agreement with the biodistribution data ( Figure 4). Radioactivity cleared from the injection site and localized in the popliteal in a high percentage, allowing for precise localization and imaging. The uptake in a second and a third lymph node observed in imaging and biodistribution studies may be due to the saturation of the mannose receptors in the small popliteal lymph node of the mouse (1-2 mg) by the excess (4.5 × 10 −13 moles) of non-radiolabeled D75CM. The latter was more noticeable in imaging studies than biodistribution because higher excess of non-radiolabeled D75CM was injected (1.8 × 10 −11 moles). Previous studies with the 99m Tc(CO) 3 -DCM20 in rats have shown that the leak to the second lymph node is eliminated by reducing the injected non-radiolabeled compound [24]. Thus, in larger animals or humans, the visualization of the SLN only is expected because the size of the lymph node and the number of mannose receptors will be higher. The marginal uptake in the lymph nodes after injecting the non-mannosylated compound 99m Tc-D75C ( Figure 5) in the footpad and the accumulation of radioactivity in the liver after intravenous injection of 99m Tc-D75CM in mice (Figure 6) strongly support the interaction of 99m Tc-D75CM with mannose receptors.
Overall, the attractive biological features of 99m Tc-D75CM, in combination with the high radiochemical yield and stability of the compound, justify its further evaluation as a new radiopharmaceutical for sentinel lymph node detection.
The ultrafiltration was performed into an ultrafiltration cell (Model 8400, Millipore Corp, Bedford, MA, USA) fitted with an ultrafiltration membrane (YM03, MW cut off 3000).
The NMR spectra were recorded in D 2 O at 25 • C on a Bruker 500 MHz Avance DRX (Bruker, Billerica, MA, USA) using sodium trimethylsilylpropanesulfonate (DSS) as an internal standard. Assignment of the spectra was based on a series of 1 H-1 H experiments described in detail in the literature [23].
HPLC analysis was performed on a Waters 600 chromatography system (Waters, Milford, MA, USA) coupled to a Waters 2487 Dual λ absorbance detector (Waters, Milford, MA, USA) and a Gabi gamma detector (Raytest, Germany). Separations were achieved on a Macherey-Nagel Nucleosil RP-C18 column (10 µm, 250 × 4 mm) eluted with a binary gradient system at a 1 mL/min flow rate. Mobile phase A was water containing 0.1% trifluoroacetic acid (TFA), while mobile phase B was methanol containing 0.1% TFA. The elution gradient was 0-1 min 95% A (5% B), followed by a linear gradient to 30% A (70% B) in 9 min; this composition was held for another 10 min.
Caution! Technetium-99m is radioactive, and all manipulations utilizing radioactive material were performed by authorized personnel, followed appropriate radiation safety procedures, and were conducted in supervised laboratories licensed for such work. Na 99m TcO 4 was obtained in physiological saline as a commercial 99 Mo/ 99m Tc generator eluate (Ultra-Technekow™ V4 Generator, Curium Pharma, Petten, The Netherlands). The radioactive precursor fac-[ 99m Tc(CO) 3 (H 2 O) 3 ] + was prepared using a homemade kit containing 5.5 mg of NaBH 4 , 4 mg of Na 2 CO 3 , and 20 mg of Na-K tartrate, purged with CO gas before the addition of Na 99m TcO 4 , as described in the literature [28].

Synthesis of Allyl-Dextran Compounds (D10A-D500A)
General method: Dextran (15.0 g), 4.0 g (1 × 10 −1 mol) of NaOH, and 0.1 g (2.6 × 10 −4 mol) of NaBH 4 were dissolved in 75 mL of distilled water. The solution was warmed to 50 • C, and allyl bromide (25.4 g, 0.21 mol) was added. The pH was maintained at 11 by the addition of 2.5 N NaOH. After 4 h, the solution was neutralized (pH 7.0) with glacial acetic acid, and the allyl-dextran was purified by precipitation with ethanol. Further purification was performed by ultrafiltration. The white solid was dissolved in 50 mL water and filtered through a 5 µm filter. The filtrate was transferred into an ultrafiltration cell, and the volume was fixed to 250 mL with water and then concentrated to 15 mL by applying N 2 gas pressure directly to the ultrafiltration cell. The retentate was diluted with 250 mL water, reconcentrated to 10 mL, and finally lyophilized. Yields ranged between 76% and 82%. All products give the same NMR peaks in D 2 O. 1

Synthesis of Dextran-S-Cysteine Compounds (D10C-D500C)
General method: To a solution of 2.0 g of allyl dextran in 10 mL of water, 1.43 g (8.14 × 10 −3 mol) L-cysteine hydrochloride monohydrate and 0.12 g (5.25 × 10 −4 mol) of ammonium persulfate ((NH 4 ) 2 S 2 O 8 ) were added, and the resulting solution was stirred for 4 h at 50 • C under nitrogen. The pH was adjusted to 4.0 using 0.1 N NaOH, and the solution was left under stirring at room temperature for 24 h. The volume was fixed to 50 mL with 0.02 M NaOAc buffer pH 4.0, and after filtration through a 5 µm filter, the filtrate was transferred into an ultrafiltration cell. The volume was fixed to 250 mL with 0.02 M NaOAc buffer pH 4.0 and then concentrated to 10 mL by applying N 2 gas pressure directly to the ultrafiltration cell. Subsequently, the retentate was diluted with 250 mL 0.1 M NaHCO 3 buffer, concentrated to 10 mL, as above; the retentate was diluted with 250 mL of water, reconcentrated to 10 mL, and lyophilized. Yields ranged between 70% and 75%. All products give the same NMR peaks in D 2 O. 1

Synthesis of Mannosylated Dextran-S-Cysteine Compounds (D10CM-D500CM)
General method: To a methanolic suspension of cyanomethyl 2,3,4,6-tetra-O-acetyl-1thio-β-D-mannopyranoside (1.41 g, 3.49 × 10 −3 mol, in 33 mL methanol), 2 mL of sodium methanoxide solution (21.6 mg, 3.99 × 10 −4 mol) was added, and the mixture was agitated periodically. After 24 h, 15 mL of the solution was transferred to a dried recovery flask, and methanol was removed by rotary evaporation, affording 2-imino-2-methoxyethyl-1-thioβ-D-mannopyranoside as golden syrup. Immediately, a solution of DC (0.2 g) in 7.5 mL of 0.02 M sodium borate buffer pH 9.0 was added to the flask and left to react for 20 h under periodic stirring. After filtration through a 5 µm filter, the filtrate was transferred into an ultrafiltration cell. The volume was fixed to 50 mL with 0.1 M NaHCO 3 buffer and then concentrated to 5 mL by applying N 2 gas pressure directly to the ultrafiltration cell. Subsequently, the retentate was diluted with 50 mL deionized water, concentrated to 5 mL as above (twice), and lyophilized. Yields ranged between 80% and 85%. All products give the same NMR peaks in D 2 O, differing in relative intensities between derivatives. 1

In Vitro Stability Studies of 99m Tc Complexes
Aliquots of 400 µL (37 MBq) of 99m Tc complexes were added to 100 µL of histidine or cysteine (5 × 10 −2 M) solution in PBS, pH 7.4, respectively. The samples were incubated for 1, 3, and 6 h, and aliquots were analyzed by HPLC.

Animal Distribution Studies
All the biodistribution studies were carried out in compliance with the Presidential Decree 56/2013 (published in the Official Government Gazette of Greece 106 A/30-4-2013) that has transposed the EU Directive 2010/63 on the protection of animals used for scientific purposes.
Groups of healthy Swiss Albino mice (male, 25 ± 3 g) under slight ether anesthesia were injected subcutaneously in the rear footpad with 99m Tc-labeled dextran derivatives. The preparation mixture of the 99m Tc-labeled compounds was diluted with saline before injection to adjust concentration. A volume of 0.02 mL (0.074 MBq, 0.05 µg DCM) was administered in three groups of three mice each to the rear footpad, while 5 min prior to sacrifice, 0.02 mL of patent blue V was also injected to facilitate lymph node visualization. After each injection, the pad was massaged for 0.5 min. At preset time intervals postinjection (15, 60, and 180 min), mice were sacrificed by cardiectomy. Lymph nodes (popliteal and inguinal) were extracted first. Other organs and samples of blood and muscle were also collected, weighed, and assayed for radioactivity. Total urine volume was collected during the experiment and added to that removed from the bladder after sacrifice. Bladder and excreted urine were not weighed. The stomach and intestines were not emptied of food contents prior to radioactivity measurements. The percentage of injected dose per organ (% ID/organ) was calculated by comparing sample radioactivity with standard solutions containing 10% of the injected dose. The calculation for blood and muscle was based on measured activity, sample weight, and body composition data (considering that blood and muscle comprise 7% and 43% of body weight). The percentage of injected dose per gram (% ID/g) was calculated by dividing the % ID/organ by the organ or tissue's weight.

Imaging Systems
Real-time, fast, dynamic screening studies were performed on a dedicated benchtop, mouse-sized, planar scintigraphy system (γ-eye TM by BIOEMTECH, Athens, Greece). The system supports fusion with a digital mouse photograph. In addition, to further enhance anatomical mouse mapping to the corresponding functional information, a deep neural network was used to translate the photographic image to an artificially produced X-ray scan for anatomical co-registration [29]. The detector is based on position-sensitive photomultiplier tubes (PSPMTs), coupled to a CsI(Na) pixelated scintillator and a mediumenergy lead collimator with parallel hexagonal holes, supporting a wide range of SPECT isotopes. The system's field of view is 5 × 10 cm 2 , with a spatial resolution of~2 mm.
For the planar imaging, healthy Swiss Albino mice were kept under isoflurane anesthesia and constant temperature of 37 • C. Short static scans were possible at different time points to provide longitudinal information on the distribution on the same animal after short anesthesia times, e.g., 10 min or less [30].
Tomographic SPECT/CT imaging was performed with y-CUBE TM and x-CUBE TM (Molecubes, Belgium). The y-CUBE TM (Molecubes, Belgium) system provides SPECT images with a spatial resolution of 0.6 mm for mouse imaging, and the accompanying x-CUBE TM (Molecubes, Belgium) can provide CT images with 50 µm resolution, operating between 35 and 80 kVp, 10 and 500 µA tube current.
Mouse imaging on the scintigraphy system was performed by keeping the mice anesthetized under isoflurane and constant temperature of 37 • C. Dynamic scans were acquired with a 2 min frame with a total of 30 min duration, based on the injected activity and system specifications.
Mouse imaging on tomographic systems was performed by keeping the mice anesthetized under isoflurane and constant temperature of 37 • C. SPECT scans were acquired with a 30-50 min duration, based on the injected activity and system specifications.
A CT scan followed each SPECT scan for co-registration purposes. The SPECT data for the Molecubes system were reconstructed through an MLEM algorithm, with 250 um voxel size and 500 iterations. CT data were reconstructed through an ISRA algorithm, with 100 µm voxel size.

Animal Imaging Studies
For SPECT isotopes, studies with 99m Tc-complexes were analyzed. In one study, intravenous bolus injections of 99m Tc-D75CM (20 µL,~10 MBq,~2 µg D75CM) were performed. In all the other studies, subcutaneous injection in the footpad of lymph node targeting agent 99m Tc-D75CM or the non-mannosylated compound 99m Tc-D75C (20 µL, 10 MBq, 2 µg D75C or D75CM) was performed. Mice were kept anesthetized during administration and imaging with isoflurane anesthesia (induction with 3-5% isoflurane flow rate and maintenance with 1-3% flow rate). The animals were imaged alive, and the studies started with 2D imaging, and the 3D scans followed right after.
For the live dynamic imaging performed with γ-eye TM , post-processing and quantification were performed through embedded analysis software, visual|eyes (BIOEMTECH, Athens, Greece).
For the tomographic images acquired with the tomographic imaging systems, postprocessing was performed through third-party analysis software, VivoQuant v1.23 (Invicro LLC, Boston, MA, USA).

Conclusions
With the aim of developing the ideal radiopharmaceutical for SLND, we worked on the synthesis, labeling, and biological evaluation of a series of mannosylated dextran derivatives. Synthesis of all derivatives was successfully achieved by a multistep procedure. Labeling with 99m Tc using the fac-[ 99m Tc(CO) 3 (H 2 O) 3 ] + synthon resulted in each case in a single product with high radiochemical purity, and the labeled products were stable.
Biodistribution studies show that all compounds have similar or better characteristics to our previously reported compound 99m Tc(CO) 3 -DCM20. In addition, compound 99m Tc-D75CM is superior to 99m Tc(CO) 3 -DCM20, as the lymph node uptake is almost 2 times higher, making it a candidate for further evaluation in larger animals and clinical trials as a new radiopharmaceutical for sentinel lymph node detection.