Introduction

Technetium-99m (99mTc) is one of the major radioisotopes widely used for clinical single-photon emission computed tomography (SPECT) imaging because it emits gamma rays with a photon energy of 141-keV, which is suitable for detection by the SPECT scanner, and it is easily produced by a molybdenum-99/technetium-99m generator1. To radiolabel 99mTc, many chelating moieties have been developed and used widely2,3,4.

Hydroxamamide (Ham) is a thiol-free chelating agent of oxo-technetium(V) [Tc(V)] known to produce 99mTc-complexes with a high radiochemical yield under moderate reaction conditions5,6,7,8. In addition, Ham makes it possible to easily produce 99mTc-labeled compounds with a bivalent targeting scaffold because it forms 99mTc-complexes with a metal-to-ligand ratio of 1:27. Our group previously developed Ham-based 99mTc radiolabeled SPECT imaging probes and demonstrated their properties for detecting their target biomolecules9,10,11,12,13. In these probes, the Ham group was directly incorporated into their chemical structures, that is to say, the reaction for induction of Ham group was not independent on the synthesis of those probes in most cases. Therefore, to develop novel Ham-based 99mTc radiolabeled probes with other targeting ligands, synthetic routes for the probes must be redesigned and established, which makes them difficult to produce. Bifunctional chelating agents composed of a metal binding moiety and a chemically reactive functional group simplify the introduction of chelating moieties to the targeting ligands14. However, no Ham-based bifunctional chelating agent for producing 99mTc-labeled bivalent ligand probes has been reported.

In this study, we designed and synthesized a novel Ham-based bifunctional chelating agent “Ham-Mal” to simplify the development of Ham-based SPECT imaging probes (Fig. 1). As a chemically reactive functional group, we chose the maleimide group, which can be easily reacted with thiol groups of ligands via Thio-Michael addition under moderate conditions15. To estimate the conditions of the 99mTc radiolabeling reaction, we synthesized “Ham-Cys”, which was acquired by the reaction with Ham-Mal and cysteine, and then performed the 99mTc radiolabeling reaction to produce “99mTc-(Ham-Cys)2” under several reaction conditions. In addition, to evaluate the utility of Ham-Mal as a 99mTc-bifunctional chelating agent, we synthesized “99mTc-(Ham-RGD)2” whose precursor, “Ham-RGD”, was acquired by reacting Ham-Mal with c(RGDfC), a cyclic arginine-glycine-aspartic acid (RGD) peptide with a high affinity to Integrin ɑvβ3, a member of the integrin superfamily of adhesion molecules16.

Figure 1
figure 1

Chemical structure of Ham-Mal.

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Results

Chemistry

The synthesis of Ham-Mal, Ham-Cys, and Ham-RGD is outlined in Fig. 2. Ham-Mal was prepared in four steps from 4-aminobenzonitrile. First, 4-aminobenzonitrile was reacted with maleic anhydride to acquire 1 and then its maleimide group was protected by furan to produce 2. A Ham group was then introduced into 2 by reacting with hydroxylamine to produce 3. Next, Ham-Mal (4) was acquired by deprotecting the maleimide group of 3, with a total yield of 7.2% from 4-aminobenzonitrile. Ham-Cys and Ham-RGD were acquired by reacting Ham-Mal (4) with cysteine and c(RGDfC), with yields of 23% and 64%, respectively.

Figure 2
figure 2

Synthetic route of Ham-Mal, Ham-Cys, and Ham-RGD.

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Radiolabeling

The 99mTc labeling of Ham-Cys was performed by reacting it with 99mTc pertechnetate and tin(II) tartrate hydrate as a reducing agent in a solvent of acetic acid/ethanol (1/4) or phosphate buffer (pH 7.4), with different final concentrations of Ham-Cys (10 or 20 mM) (Fig. 3). The radiochemical yields of 99mTc-(Ham-Cys)2 reacted in phosphate buffer were markedly higher than those in acetic acid/ethanol solvent [phosphate buffer: 68.7 ± 2.4% (final concentration of Ham-Cys: 10 mM), 75.3 ± 4.5% (20 mM), acetic acid/ethanol solvent: 10.4 ± 9.8% (10 mM), 11.0 ± 5.0% (20 mM)] (Table 1). On the other hand, the concentration of the precursor (Ham-Cys) did not affect the radiochemical yield (Table 1). 99mTc-(Ham-RGD)2 was obtained by reacting Ham-RGD (final concentration: 10 mM) with 99mTc pertechnetate and tin(II) tartrate hydrate as a reducing agent in the solvent of phosphate buffer (pH 7.4), with a radiochemical yield of a 49.0 ± 4.8% and radiochemical purity of over 95% (Fig. 4). We also confirmed the purified 99mTc-(Ham-Cys)2 and 99mTc-(Ham-RGD)2 did not contained the respective precursor (Ham-Cys and Ham-RGD, respectively) (Supplementary file, Figure S1).

Figure 3
figure 3

99mTc labeling route of 99mTc-(Ham-Cys)2 and 99mTc-(Ham-RGD)2.

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Table 1 Radiochemical yields of 99mTc-(Ham-Cys)2 under each condition.
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Figure 4
figure 4

Radiochromatograms for 99mTc-(Ham-Cys)2 (A) and 99mTc-(Ham-RGD)2 (B).

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In vitro cellular uptake study

The cellular uptake of 99mTc-(Ham-RGD)2 is expressed as the %dose/mg protein (Fig. 5). The radioactivity of U87MG cells was significantly higher than that of PC3 cells (U87MG: 1.87 ± 0.28 vs. PC3: 0.49 ± 0.08%dose/mg protein, p < 0.01). In addition, the radioactivity of U87MG cells was significantly reduced by pretreatment with excess c(RGDfK) (0.95 ± 0.04%dose/mg protein, p < 0.01) (Fig. 5).

Figure 5
figure 5

In vitro cellular binding of 99mTc-(Ham-RGD)2 in U87MG and PC3 cells. Results are expressed as the mean ± standard error of six independent experiments.

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In vitro stability of 99mTc-(Ham-RGD)2 in mouse plasma

The in vitro stability of 99mTc-(Ham-RGD)2 in mouse plasma was evaluated by incubating the probe in murine plasma at 37 °C for 60 and 180 min. The radiochemical purity of 99mTc-(Ham-RGD)2 was 37.3 ± 3.7% (60 min) and 15.7 ± 5.8% (180 min), respectively.

In vivo biodistribution

The uptake of 99mTc-(Ham-RGD)2 in each organ was expressed as the %ID/g, except for that in the stomach and thyroid, which was expressed as the %ID (Table 2). 99mTc-(Ham-RGD)2 was excreted mainly from the kidneys. The radioactivity of U87MG tumors (high Integrin ɑvβ3 expression) was significantly higher than that of PC3 tumors from 5 min after the administration of 99mTc-(Ham-RGD)2. In addition, the accumulation in U87MG tumors was reduced by blocking with excess c(RGDfK) at 180 min after administration [6.75 ± 1.73%ID/g (non-blocking) vs. 4.00 ± 0.68%ID/g (blocking) (p < 0.01)], but there was no significant difference in accumulation in PC3 tumors between the non-blocking and blocking groups [1.30 ± 0.35%ID/g (non-blocking) vs. 1.29 ± 0.12%ID/g (blocking) (p = 1.00)] (Supplementary file, Table S1).

Table 2 Biodistribution of 99mTc-(Ham-RGD)2 in the U87MG/PC3 tumor-bearing mice.
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SPECT imaging

The SPECT/CT images were acquired at 60 and 180 min after the administration of 99mTc-(Ham-RGD)2 (Fig. 6). The U87MG tumor was clearly visualized from 60 min, whereas the radioactive signal in the PC3 tumor was limited.

Figure 6
figure 6

SPECT/CT images (upper: axial, lower: coronal) of the U87MG/PC3 tumor-bearing mouse after the administration of 99mTc-(Ham-RGD)2. 99mTc-(Ham-RGD)2 (2.8 MBq/0.1 mL saline) was injected intravenously into the mouse, and the SPECT imaging was acquired at 60 (left) and 180 min (right) after the administration. The CT images were acquired just before the SPECT imaging. The Yellow circle: U87MG tumor, white circle: PC3 tumor.

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Discussion

In this study, we designed and synthesized “Ham-Mal” as a novel Ham-based bifunctional chelating agent for 99mTc. Ham-Mal was obtained through a four-step reaction from 4-aminobenzonitrile with a total yield of 7.2% (Fig. 2). We first converted the cyanide group of 1 to the Ham group directly; however, this reaction failed, possibly due to the instability of the maleimide group of 1. Maleimide groups can be protected and deprotected with furan by the thermally reversible Diels–Alder click reaction17. Thus, the Ham group was introduced after protecting the maleimide group of 1. After the synthesis of Ham-Mal, we conjugated ligands [cysteine and c(RGDfC)] having a thiol group with Ham-Mal via the thiol-maleimide reaction, and obtained Ham-Cys and Ham-RGD, respectively (Fig. 2).

Next, we performed the radiolabeling of Ham-Cys and Ham-RGD with 99mTcO4-. In general, the Ham group structure is known to have two tautomer, and thus the 99mTc-Ham complex forms two tautomer which can be observed by RP-HPLC 7. The chelation of Ham and 99mTc was confirmed by the disappearance of the peak from 99mTcO4- (retention time: 2–4 min11) and then the appearance of two peaks because it is difficult to synthesize the analogues of the rhenium-Ham complex7. To search for an appropriate 99mTc radiolabeling reaction condition, we first evaluated the reaction of Ham-Cys and 99mTcO4- with different concentrations of Ham-Cys (final concentration: 10 and 20 mM) in different solvents [phosphate buffer (pH 7.4) and acetic acid/ethanol = 1/4]. In our previous study, we used an acetic acid/ethanol mixture as the solvent for 99mTc radiolabeling reactions of compounds containing Ham9,10,11,12,13 to prevent forming by-products (especially, 99mTc-colloid); however, the radiochemical yields were low [10.4 ± 9.8% (final concentration of Ham-Cys:10 mM), 11.0 ± 5.0% (20 mM)] (Table1) and the acquired 99mTc-radiolabeled compound was unstable. On the other hand, 99mTc radiolabeling reaction to give 99mTc-(Ham-Cys)2 in phosphate buffer (pH 7.4) proceeded with radiochemical yields of 68.7 ± 2.4% (final concentration of Ham-Cys: 10 mM) and 75.3 ± 4.5% (20 mM) (Table 1). The acquired 99mTc-radiolabeled compound was analyzed by RP-HPLC and only two radioactive peaks (retention times: 19.2 min and 20.6 min) were observed (Fig. 4A). Therefore, this suggested that the 99mTc-radiolabeling reaction of compounds based on Ham-Mal proceeded in the neutral pH solvent. As for the concentration of the precursor, a final concentration of 10 mM was considered sufficient for the 99mTc-radiolabeling reaction (Table 1). Thus, we performed the radiolabeling of Ham-RGD with 99mTc using 10 mM (final concentration) Ham-RGD in phosphate buffer (pH 7.4), and acquired 99mTc-(Ham-RGD)2 in a radiochemical yield of a 47% with a radiochemical purity of over 95% (Fig. 4B).

To confirm that 99mTc-radiolabeled agents composed of Ham-Mal are able to target the biomolecules to which the ligands conjugated to Ham-Mal have a high affinity, we next performed in vitro and in vivo studies with 99mTc-(Ham-RGD)2, and evaluated it as an Integrin ɑvβ3-targeting probe. Integrin ɑvβ3 is a well-known biomolecule that is closely related to the neovasculature and metastasis of cancer18. In addition, c(RGDfC), a targeting ligand of 99mTc-(Ham-RGD)2, is known to have a high affinity to Integrin ɑvβ319. In the in vitro study, the cellular uptake level of 99mTc-(Ham-RGD)2 in U87MG cells (high Integrin αvβ3 expression) was significantly higher than that in PC3 cells (low Integrin ɑvβ3 expression) (U87MG: 1.87 ± 0.28 vs. PC3: 0.49 ± 0.08%dose/mg protein, p < 0.01) (Fig. 5). In addition, the uptake level of 99mTc-(Ham-RGD)2 in U87MG cells was significantly reduced by pretreatment with excess non-radiolabeled cyclic RGD peptide [c(RGDfK)] (0.95 ± 0.04%dose/mg protein, p < 0.05) (Fig. 5). This suggests that 99mTc-(Ham-RGD)2 was taken up specifically by cells with high Integrin ɑvβ3 expression.

In the in vivo biodistribution study, 99mTc-(Ham-RGD)2 also accumulated highly in U87MG tumors compared with PC3 tumors from 5 min after its i.v. administration (Table 2). In addition, the accumulation level in U87MG tumors was reduced by blocking with excess non-radiolabeled c(RGDfK) at 180 min after administration [4.00 ± 0.68%ID/g (blocking) (p < 0.05)] (Supplementary file, Table S1), which suggests that 99mTc-(Ham-RGD)2 accumulated specifically in tumors with high Integrin ɑvβ3 expression. We also performed SPECT/CT imaging study of 99mTc-(Ham-RGD)2, and confirmed that 99mTc-(Ham-RGD)2 can selectively visualize U87MG tumors, consistent with the in vivo biodistribution study (Fig. 6). These results support the use of 99mTc-(Ham-RGD)2 as a SPECT imaging probe targeting tumors with high Integrin ɑvβ3 expression. In the in vivo biodistribution study, we also observed high radioactivity in the kidneys (Table 2). This may be due to the properties of c(RGDfC) because Integrin ɑvβ3 was reported to be expressed in the kidneys, especially in glomerular epithelial cells and Bowman's capsule20, and that the conventional agents based on RGD peptides accumulated highly in the kidneys21. As for the stomach and thyroid where free 99mTc (99mTcO4−) is known to accumulate highly22, less radioactivity was observed at 360 min after the administration of 99mTc-(Ham-RGD)2 (Table 2). We also evaluated the in vitro stability of 99mTc-(Ham-RGD)2 in mouse plasma; however, we found that the radiochemical purity decreased in a time dependent manner, and some unknown hydrophilic metabolites emerged (Figure S2). Given that other Ham-based 99mTc radiolabeled compounds we developed previously11,13 showed high stability in plasma and good blood clearance, it is suspected that those unknown metabolites affected the slow blood clearance. Although the metabolites and the reason of the instability is still unclear, the low stability of 99mTc-(Ham-RGD)2 in plasma is the limitation, and further study for redesigning the bifunctional chelate to improve the stability would be needed for use it in in vivo study.

To date, several chelating groups, such as D-penicillamine23,24 and isocyanide25,26, have been reported to form multivalent complexes with 99mTc. These chelates reacted with 99mTc effectively at a low concentration of the precursors, but their reaction temperature was high (100–120 °C). On the other hand, the precursors based on Ham-Mal reacted with 99mTc under moderate conditions (at room temperature), which is suitable for radiolabeling thermally instable compounds such as proteins (e.g. antibody and its fragment). For radiolabeling 99mTc with those thermally instable compounds, some chelates such as hydrazinonicotinamide (HYNIC) have been used27,28. Given that these chelates form 99mTc-complexes with a metal-to-ligand ratio of 1:1, Ham has an advantage in terms of developing 99mTc-labeled compounds with a bivalent targeting scaffold, so that Ham-Mal would be a promising bifunctional chelate for developing those 99mTc-labeled compounds.

In this study, we designed and synthesized a novel bifunctional 99mTc-chelating agent based on Ham, “Ham-Mal”. 99mTc radiolabeled agents were acquired easily by reacting Ham-Mal and the targeting ligands via thiol-maleimide reaction, and then by the 99mTc-radiolabeling reaction under moderate conditions. The acquired 99mTc radiolabeled agent [99mTc-(Ham-RGD)2 in this study]demonstrated good visualization of specific organs expressing the target biomolecule. This study suggests that Ham-Mal is available for the development of 99mTc-radiolabeled SPECT imaging probes containing bivalent targeting ligands.

Methods

Materials

All reagents were obtained commercially and used without further purification unless otherwise indicated. 99mTc-NaTcO4 was purchased from Nihon Medi-Physics Co., Ltd. (Tokyo, Japan). W-Prep 2XY (Yamazen, Osaka, Japan) was used for silica gel column chromatography on a Hi Flash silica gel column (40 μm, 60 Å, Yamazen). 1H NMR and 13C NMR spectra were recorded on a JNM-ECS400 (JEOL, Tokyo, Japan) with tetramethylsilane as an internal standard. Coupling constants are reported in Hertz. Multiplicity was defined as singlet (s), doublet (d), or multiplet (m). High-resolution mass spectrometry (HRMS) of fast atom bombardment (FAB) and electrospray ionization (ESI) were carried out with a JEOL JMS-700 (JEOL) and a LCMS-IT-TOF (SHIMADZU, Kyoto, Japan), respectively. High-performance liquid chromatography (HPLC) was performed with a Shimadzu system (SHIMADZU, an LC-20AT pump with an SPD-20A UV detector, λ = 254 nm).

Chemistry

Synthesis of 1

A solution of 4-aminobenzonitrile (2.36 g, 20 mmol) and maleic anhydride (5.88 g, 60 mmol) in 1,4-dioxane (30 mL) was stirred at 100 °C for 1 h. To the reaction mixture, ammonium persulfate (9.12 g, 40 mmol) and dimethyl sulfoxide (DMSO) (2.84 mL) were added and the reaction mixture was heated to 100 °C for 3 h. Thereafter, the reaction mixture was filtered and dioxane was removed under vacuo. The residue was dissolved in ethyl acetate and washed with diluted HCl, saturated aqueous NaHCO3, and brine. The organic layer was dried over Na2SO4 and the solvent was evaporated under vacuo to afford 3.38 g of 1 (yield: 85%). 1H NMR (400 MHz, DMSO-d6) δ 7.25 (s, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 109.88, 118.49, 126.86, 133.06, 135.02, 135.88, 169.34.

Synthesis of 2

A solution of 1 (3.31 g, 16.7 mmol) in acetonitrile (10 mL) was mixed with furan (5 mL) and stirred at 60 °C for 8 h. The solvent was evaporated and ethyl acetate was added, then a solid precipitated out. The material was filtered and washed with ethyl acetate. The filtrated solution was concentrated to afford more of the product, which was also filtered and washed with ethyl acetate. The combined solid portions were dried under high vacuum at room temperature overnight to afford 2.67 g of 2 (yield: 60%). 1H NMR (400 MHz, DMSO-d6) δ 3.13 (s, 2H), 5.26 (s, 2H), 6.62 (s, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 47.71, 80.91, 110.98, 118.29, 127.55, 133.19, 136.05, 136.72, 175.27. HR-MS (FAB, pos) m/z calcd. for C15H11N2O3+, 267.0770; found 267.0768 [M + H]+.

Synthesis of 3

A solution of hydroxylamine hydrochloride (521 mg, 7.5 mmol) and triethylamine (1.04 mL, 7.5 mmol) in N,N-dimethylformamide (DMF) (5 mL) and H2O (2 mL) was added to a solution of 2 (1.33 g, 5 mmol) in DMF (10 mL). The reaction mixture was stirred at room temperature for 3 h. Then, H2O was added to the solution and diluted with ethyl acetate. The organic layer was dried over Na2SO4 and the solvent was evaporated under vacuo. The residue was recrystallized with chloroform and dried under high vacuum at room temperature overnight to afford 287 mg of 3 (yield: 19%). 1H NMR (400 MHz, DMSO-d6) δ 3.08 (s, 2H), 5.24 (s, 2H), 5.88 (s, 2H), 6.60 (s, 2H), 7.19 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 9.74 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 47.51, 80.80, 126.01, 126.46, 132.41, 133.41, 136.66, 150.27, 175.68. HR-MS (FAB, pos) m/z calculated for C15H14N3O4+, 300.0984; found 300.0985 [M + H]+.

Synthesis of 4 (Ham-Mal)

A solution of 3 (299 mg, 1 mmol) in DMF (6 mL) and toluene (18 mL) was stirred at 115 ºC for 2 h. After evaporation of the solvent, the residue was suspended in H2O and then filtered. The solid portion was dried under high vacuum at room temperature overnight to afford 231 mg of 4 (yield: 74%). 1H NMR (400 MHz, DMSO-d6) δ 5.87 (s, 2H), 7.20 (s, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.8 Hz, 2H), 9.72 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 125.85, 126.28, 131.94, 132.65, 134.73, 150.29, 169.82. HR-MS (ESI, pos) m/z calculated for C11H10N3O3+, 232.0722; found 232.0725 [M + H]+.

Synthesis of 5 (Ham-Cys)

A solution of L-cysteine (10 mg, 80 µmol) in H2O (100 µL) was adjusted to pH 7–8 by 0.1 M NaOH and then added to a solution of 4 (10 mg, 40 µmol) in DMF (500 µL). The reaction mixture was stirred at room temperature for 1 h. Then, the solvent was evaporated and the residue was purified by RP-HPLC on a Cosmosil 5C18-AR-II column (Nacalai Tesque, Kyoto, Japan, diameter: 10 mm, length: 250 mm, particle size: 5 μm, pore size: 120 Å) using the mobile phase [H2O (0.1% TFA)/acetonitrile (0.1% TFA) = 90/10] at a flow rate of 4.0 mL/min to give 3.5 mg of 5 (yield: 23%). 1H NMR (400 MHz, D2O) δ 3.07–3.14 (m, 2H), 3.24–3.34 (m, 2H), 4.08–4.18 (m, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H). ESI-HRMS m/z calculated for C14H17N4O5S+, 353.0920; found, 353.0919 [M + H]+.

Synthesis of 6 (Ham-RGD)

A solution of c(RGDfC) (PEPTIDE INSTITUTE, INC., Osaka, Japan, 2.0 mg, 4 µmol) in H2O (200 µL) was added to a solution of 4 (0.8 mg, 4 µmol) in DMF (500 µL). The reaction mixture was stirred at room temperature for 1 h. Then, the solvent was evaporated and the residue was purified by RP-HPLC on a Cosmosil 5C18-AR-II column (Nacalai Tesque, diameter: 10 mm, length: 250 mm, particle size: 5 μm, pore size: 120 Å) using the mobile phase [H2O (0.1% TFA)/acetonitrile (0.1% TFA) = 90/10 (0 min) to 70/30 (30 min)] at a flow rate of 4.0 mL/min to give 1.8 mg of 6 (64% yield). HR-MS (ESI, pos) m/z calculated 810.2993, found 810.2988 [M + H]+.

Radiolabeling of 99mTc-(Ham-Cys)2

To a solution of 5 (Ham-Cys, final concentration: 10 mM or 20 mM) in an acetic acid/ethanol mixture (1/4, 50 μL) or phosphate buffer (pH 7.4, 10 mM, 50 µL), 100 μL of 99mTc-NaTcO4 solution (18–30 MBq) and 15 μL of 3 mM tin(II) tartrate hydrate solution in H2O were added. The reaction mixture was incubated at room temperature for 10 min and purified by RP-HPLC on a YMC-Triart C18 (YMC CO., LTD., Kyoto, Japan, diameter: 4.6 mm, length: 150 mm, particle size: 5 μm, pore size: 120 Å) using the mobile phase [10 mM phosphate buffer (pH 7.4)/acetonitrile = 100/0 (0 min) to 70/30 (30 min)] at a flow rate of 1.0 mL/min. The two peaks (retention time: around 19 to 20 min, and around 20 to 21 min) were collected, mixed, and used as 99mTc-(Ham-Cys)2 in the following study. The radiochemical yield of 99mTc-(Ham-Cys)2 was calculated by dividing the radioactivity of the collected two peaks by the radioactivity of the reaction mixture measured just before the HPLC purification, in accordance with our previous study11. The acquired 99mTc-(Ham-Cys)2 was analyzed by RP-HPLC using the same method described above. The radiochemical purity was calculated by dividing the radioactivity of the two peaks derived from the 99mTc-(Ham-Cys)2 by the total radioactivity acquired from the HPLC chart (Fig. 4A).

Radiolabeling of 99mTc-(Ham-RGD)2

To a solution of 6 (Ham-RGD, final concentration: 10 mM) in phosphate buffer (pH 7.4, 10 mM, 50 µL), 100 μL of 99mTc-NaTcO4 solution (18–33 MBq) and 15 μl of 3 mM tin(II) tartrate hydrate solution in H2O were added. The reaction mixture was incubated at room temperature for 10 min and purified by RP-HPLC on a YMC-Triart C18 (YMC CO., LTD., diameter: 4.6 mm, length: 150 mm, particle size: 5 μm, pore size: 120 Å) using the mobile phase [10 mM phosphate buffer (pH 7.4)/acetonitrile = 80/20 (0 min) to 60/40 (30 min)] at a flow rate of 1.0 mL/min. The two peaks (retention time: around 13 min, and around 15 min) were collected, mixed, and used as 99mTc-(Ham- RGD)2 in the following study. The radiochemical yield of 99mTc-(Ham-RGD)2 was calculated by dividing the radioactivity of the collected two peaks by the radioactivity of the reaction mixture measured just before the HPLC purification, in accordance with our previous study11. The acquired 99mTc-(Ham-RGD)2 was analyzed by RP-HPLC using the same method described above. The radiochemical purity was calculated by dividing the radioactivity of the two peaks derived from the 99mTc-(Ham- RGD)2 by the total radioactivity acquired from the HPLC chart (Fig. 4B).

Cellular uptake study of 99mTc-(Ham-RGD)2

According to the previously reported study, U87MG human glioblastoma cells (DS Pharma Biomedical, Osaka, Japan) and PC3 human prostate carcinoma cells (DS Pharma Biomedical) were used as Integrin ɑvβ3 high- and low-expressing cells, respectively29. U87MG and PC3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) or Roswell Park Memorial Institute 1640 (RPMI 1640) containing 10% fetal bovine serum, and 100 U/mL of penicillin and streptomycin in a 5% CO2 incubator at 37 ºC. These cells were sub-cultured overnight in 12-well plates (4 × 105 cells/1 mL), and then pre-incubated in DMEM (1 mL) with or without c(RGDfK) (final concentration: 10 µM) for 30 min at 37 ºC in a humidified atmosphere containing 5% CO2. After the pre-incubation, the medium was exchanged with DMEM containing 99mTc-(Ham-RGD)2 (74 kBq, 1 mL) and then incubated for 60 min. After the incubation, the cells were washed twice with phosphate-buffered saline (PBS) (1 mL) and then lysed with 1 M NaOH (0.4 mL). The radioactivity in the lysates was measured by a Wallac WIZARD 2470 gamma counter (PerkinElmer, Waltham, MA, USA) and total protein concentrations in samples were measured using the bicinchonic acid method.

In vitro stability of 99mTc-(Ham-RGD)2 in mouse plasma

The in vitro stability of 99mTc-(Ham-RGD)2 in mouse plasma was performed in accordance to our previous study with some modification11. In brief, 99mTc-(Ham-RGD)2 (about 500 kBq) was added to prepared fresh murine plasma (200 μL) collected from ddY mice (male, 5 weeks old). After incubating the solution at 37 °C for 60 and 180 min, acetonitrile (200 μL) was added. Subsequently, the solution was centrifuged (4000g, 10 min) and the supernatant was analyzed using RP-HPLC on a YMC-Triart C18 (YMC CO., LTD., diameter: 4.6 mm, length: 150 mm, particle size: 5 μm, pore size: 120 Å) using the mobile phase [10 mM phosphate buffer (pH 7.4)/acetonitrile = 80/20 (0 min) to 60/40 (30 min)] at a flow rate of 1.0 mL/min.

In vivo biodistribution of 99mTc-(Ham-RGD)2

Animal experiments were approved by the Kyoto University Animal Care Committee, and performed in accordance with ARRIVE guidelines (https://arriveguidelines.org) and the institutional guidelines for animal care of Kyoto University. BALB/c athymic nude mice (male, 5 weeks) supplied by Japan SLC, Inc. (Hamamatsu, Japan) were housed under a 12-h light/12-h dark cycle, and given free access to food and water. PC3 cells [5 × 106 cells/0.1 mL of RPMI1640 and Geltrex (Thermo Fisher Scientific, Waltham, MA, USA) at a ratio of 1:1] were subcutaneously inoculated into the left flank of mice. At 2 weeks after the inoculation of PC3 cells, U87MG cells [5 × 106 cells/0.1 mL of DMEM and Geltrex (Thermo Fisher Scientific) at a ratio of 1:1] were subcutaneously inoculated into the right flank of mice, and a biodistribution study was performed after a 2-week growth period. 99mTc-(Ham-RGD)2 (370 kBq/0.1 mL saline) was injected intravenously into the tumor-bearing mice. Mice were sacrificed by decapitation at 5, 60, 180, and 360 min after administration. The tumor, blood, muscle, and other organs were then excised, and their radioactivity and weight were measured. For the blocking study, c(RGDfK) (10 mg/kg weight) dissolved in 0.1 mL of saline was injected intravenously into the tumor-bearing mice. At 15 min after the injection of c(RGDfK), 99mTc-(Ham-RGD)2 (370 kBq/0.1 mL saline) was injected intravenously into the mice, which were sacrificed by decapitation at 180 min after administration. The tumor, blood, muscle, and other organs were then excised, and their radioactivity and weight were measured.

SPECT imaging

SPECT/CT imaging study was performed using a Triumph LabPET12/SPECT4/CT (TriFoil Imaging Inc., Chatsworth, CA, USA) by the previously reported method with modification30. 99mTc-(Ham-RGD)2 (2.8 MBq/0.1 mL saline) was injected intravenously into the tumor-bearing mice prepared by the above method. At 45 and 165 min after administration, the mice were anesthetized with 1.5–2.0% isoflurane for approximately 10 min, and then CT imaging was performed with X-ray sources set to 60 kVp and 360 μA. After CT imaging at 60 and 180 min after the administration of 99mTc-(Ham-RGD)2, SPECT imaging was performed with multi-pinhole collimators (1.0 mm diameter) and the projection data were acquired using a 20% energy window centered at 140 keV for 99mTc, with a 360° circular orbit, 60-s projection time, and 32 projection angles. The acquired SPECT data were reconstructed by a three-dimensional ordered subset expectation maximization (OSEM) algorithm with CT-based attenuation correction and then analyzed using Amide software (Amide.exe ver. 1.0.4, SourceForge.net, https://sourceforge.net/projects/amide/files/amide/1.0.4/).

Statistics

Data are presented as the mean ± S.E.M (for the in vitro study) or the mean ± S.D (for the in vivo study). In the in vitro study, statistical analyses were performed using 2-way ANOVA following the Tukey–Kramer test with JMP 14 software (SAS Institute Inc., Cary, NC, USA). For the in vivo study, the Student’s t-test was used to assess the significance of differences. Differences at the 95% confidence level (P < 0.05) were considered significant.