99mTc Radiolabelling of Fe3O4-Au core-shell and Au-Fe3O4 Dumbbell-like nanoparticles

The development of nanoparticle-based dual-modality probes for magnetic resonance imaging (MRI) and positron emission tomography (PET) or single photon emission computed tomography (SPECT) is increasingly growing in importance. One of the most commonly used radionuclides for clinical SPECT imaging is (99m)Tc and the labelling of Fe3O4 nanoparticles with (99m)Tc was shown to be a successful strategy to obtain dual-modality imaging agents. In this work, we focus on gold containing magnetic nanomaterials. The radiolabelling of magnetic Fe3O4-Au core-shell and Fe3O4-Au dumbbell-like nanoparticles with the [(99m)Tc(CO)3](+) fragment is described. The key elements for this (99m)Tc labelling approach are novel coating ligands, consisting of an anchor for the Au surface, a polyethylene glycol linker and a strong chelator for the [(99m)Tc(CO)3](+) moiety.

Water was doubly distilled before use.PD-10 size exclusion columns (Sephadex G-25 medium) were purchased from GE Healthcare (Switzerland).Isolink TM kits were a gift from Mallinckrodt Medical B.V. (Netherlands).Na 99m TcO 4 in 0.9% saline was eluted from a 99 Mo/ 99m Tc UTK FM generator from Mallinckrodt Medical B.V. (Netherlands).

2) Characterisation
The UV-Vis measurements were collected on a Perkin Elmer Lambda 35 UV-Vis spectrophotometer between 200 and 900 nm. 1 H NMR and 13 C NMR spectra were recorded on a BrukerDRX 400 MHz or BrukerDRX 500 MHz spectrometer. 1 H and 13 C chemical shifts were referenced with the residual solvent resonances relative to TMS.
Electronspray-Ionisation mass spectrometry (ESI-MS) was performed on a Bruker esquire TM /LC spectrometer or on a Bruker esquire TM /HCT TM spectrometer.High-resolution mass spectrometry (HR-MS) was performed on a Thermo DFS double-focusing system (ThermoFisher Scientific, Germany).Inductively coupled plasma mass spectrometry (ICP-MS) was measured on an Agilent QQQ8800 Triple Quad, equipped with a standard x-lens setting, nickel cones and a "micro-mist" quartz nebulizer.Tune settings were based on the Agilent General Purpose method and only slightly modified by an autotune procedure.Iron was measured as isotope 56 Fe, gold as isotope 197 Au and the values are reported as the average of 50 sweeps with five replicates.Dynamic light scattering (DLS) measurements were carried out on a Malvern Zetasizer Nano Series instrument utilizing 90° backscatter at 25 °C.The concentration of stock NPs solutions were 0.5 mg/ml in phosphate buffered saline (PBS, pH 7.4), and all NPs were filtered through a 0.2 μm filter before analysis.
Typical count rates were 150-300 kHz.Hydrodynamic diameter (HDD) data are reported as the mean of triplicate measurements.Transmission electron microscopy (TEM) was done on a FEI Tecnai G2 Spirit operated at 120 kV (NP analysis after synthesis).One drop of a dilute sample of NPs in hexane or PBS was placed onto a formvar-coated copper grid, allowing the solvent to evaporate.Size analysis was performed on captured digital images using ImageJ V.1.47m,the size was determined from an average of 50 measurements and expressed as mean value ± standard deviation.Analytical HPLC was performed on a Merck L7000 system, using a Macherey-Nagel Nucleosil C18 column.HPLC solvents were 0.1% TFA (solvent A) and MeOH HPLC grade (solvent B).The HPLC gradient used is as follows: 0-3 minutes: 100% A; 3.1-9 minutes: 75% A, 25% B; 9.1-20 minutes: linear gradient from 66% A (34% B) to 0% A (100% B); 20-28 minutes: 100% B; 28.1-30: 100% A. The flow rate was 0.5 ml/min.Detection was performed at 254 nm.The system was equipped with the UV-detector L-7400 and the γ-detector Berthold FlowStar LB513.Activity measurements were carried out with a VDC-304 dose calibrator (Veenstra Instruments).Please note that 0.5 ml fractions were collected and the very last fraction was the remainder activity in the PD-10 column (including the activity in the column).The very first 1.0 ml (two 0.5 ml fractions) is not shown in any of the chromatograms since it always was mobile phase only. 1 Activity after incubation at 50 °C for 2 h; 2 Activity loaded on PD-10 column (in % of the total activity); 3 Combined activity from the fractions 0.5 -2.5 ml; 4 Radiochemical yield; 5 Control experiments, 1.5 ml 99m Tc complex loaded on PD-10.

t., 4 h OTs
Tetraethylene glycol (5.0 g, 25.7 mmol) and p-toluenesulfonyl chloride (14.7 g, 77.2 mmol) were dissolved in tetrahydrofurane (100 ml) and cooled to 0 °C.Potassium hydroxide (10.1 g, 180.2 mmol) in water (25 ml) was added dropwise over a period of 60 min.The mixture was stirred at room temperature for additional 3 h and afterwards poured in a 2:1 diethyl ether / water mixture (150 ml).The water phase was extracted with diethyl ether (2 x 100 ml).The combined ether phases were washed with brine (100 ml), dried over magnesium sulfate, filtered and concentrated under reduced pressure.The residue was purified by silica gel column chromatography (95:5 ethyl acetate / methanol) to give a colourless oil.Yield: 12.9 g (99%).
The mixture was stirred at room temperature for 5 h.The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (10:1:0.03dichloromethane / methanol / 25% ammonia solution) to give a yellow oil.Yield: 4.0 g (85%).
The combined organic phases were washed with water, brine and dried over magnesium sulfate.The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (10:1 dichloromethane / methanol) to give a yellow oil.Yield: 625 mg (70%). 1

ethyl N-[17-(1,2-dithiolan-3-yl)-13-oxo-3,6,9-trioxa-12-azaheptadec-1-yl]-N-(pyridin-2-
ylmethyl)glycinate (according to Mattoussi et al. 6 ) ylmethyl)glycinate (• 3 HCl; 538 mg, 1.1 mmol) was dissolved in dry dichloromethane (10 ml) and triethylamine (548 μl, 4.0 mmol).Afterwards the amine solution was added dropwise to the mixture with activated lipoic acid, followed by stirring at room temperature for 17 h.The reaction mixture was washed with water (2 x 20 ml) and saturated sodium carbonate solution (2 x 20 ml).The organic phase was dried over magnesium sulfate.The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (10:1 dichloromethane / methanol) to give a yellow oil.Yield: 464 mg (74%). 1   (386 mg, 0.7 mmol) was dissolved in methanol (1.5 ml) and 1 M aqueous lithium hydroxide solution (1.5 ml) was added.The mixture was stirred at room temperature for 1.5 h and neutralized with Amberlite IR120 H to reach pH 7. The suspension was filtered and the solvent was removed under reduced pressure to give a yellow oil.Yield: 337 mg (92%).Ethyl glyoxylate (50% solution in toluene, 0.59 ml, 2.9 mmol) was added to the suspension.The mixture was stirred at room temperature for 4 h and afterwards quenched with water (15 ml).The mixture was extracted with chloroform (3 x 50 ml).The combined organic phases were washed with water, brine and dried over magnesium sulfate.The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (10:1 dichloromethane / methanol) to give the product as a colourless oil.Yield: 515 mg (81%). 1   azatetradecan-14-oate (• 2 HCl; 460 mg, 1.1 mmol) was dissolved in dry dichloromethane (10 ml) and triethylamine (374 μl, 2.7 mmol).Afterwards the amine solution was added dropwise to the mixture with activated lipoic acid, followed by stirring at room temperature for 17 h.The reaction mixture was washed with water (2 x 15 ml) and saturated sodium carbonate solution (2 x 15 ml).The organic phase was dried over magnesium sulfate.The solvent was reduced under reduced pressure and the residue was purified by silica gel column chromatography (10:1 dichloromethane / methanol) to give a yellow oil.Yield: 443 mg (74%). 1   was added.The mixture was stirred at room temperature for 1.5 h and neutralized with Amberlite IR120 H to reach pH 7. The suspension was filtered and the solvent was removed under reduced pressure to give a yellow oil.Yield: 119 mg (86%).

1-cyano-2-ethoxy
The temperature was kept below 20 °C during the addition.After stirring for another 30 min, extraction with dichloromethane (4 x 60 ml) was carried out.The combined organic phases were dried with magnesium sulfate and the solvent was removed under reduced pressure.The resulting yellow oil was dried under vacuum.Yield: 6.1 g (67%).This oil was immediately diluted with diethyl ether (50 ml) and a solution of p-toluenesulfonic acid monohydrate (10.1 g, 53.1 mmol) in ethanol (30 ml) was added.After stirring at room temperature for 15 min, the mixture was diluted with diethyl ether (100 ml) to induce crystallization.The flask was kept overnight at -20 °C and the precipitated white crystals were collected via filtration.Yield: 7.8 g (37%).To a solution of di-tert-butyl dicarbonate (8.0 g, 36.6 mmol) in toluene (25 ml) was added 1-cyano-2ethoxy-2-oxoethanaminium 4-methylbenzenesulfonate (6.4 g, 20.9 mmol) and N,N-diisopropylethylamine S26 (3.6 ml, 20.9 mmol).The reaction mixture was refluxed at 100 °C for 6 h.After cooling to room temperature, water (25 ml) was added, and the resulting suspension was poured into ethyl acetate (300 ml).The organic layer was extracted with 1:1 water / saturated aqueous sodium hydrogen carbonate (200 water (200 ml) and brine (200 ml).The organic phase was dried over sodium sulfate, filtered and concentrated in vacuum.The residue was purified by silica gel column chromatography (4:1 hexane / ethyl acetate) to give the product as a white solid.Yield: 3.2 g (70%). 1 H NMR (400 MHz, CDCl 3 , ppm) δ 5.37 (s, 1H), 5.26 (d, 1H, J=7.8 Hz), 4.36 (q, 2H, J=7.2 Hz), 1.48 (s, 9H), 1.36 (t, 3H, J=7.2 Hz).R f = 0.35 (4:1 hexane / ethyl acetate).Triethylene glycol (5.5 ml, 41.3 mmol) was dissolved in 50% aqueous sodium hydroxide solution and stirred at room temperature for 10 min.Benzyl chloride (5.0 ml, 43.4 mmol) was added and the mixture was refluxed at 105 °C for 15 h.After cooling to room temperature, extraction with dichloromethane (3 x 200 ml) was carried out and the organic layer was washed with water (150 ml) and brine (150 ml).The organic phase was dried over magnesium sulfate, filtered and concentrated under reduced pressure.The residue was purified by silica gel column chromatography (ethyl acetate) to give a colourless oil.Yield:  was added to the solution and the reaction mixture was refluxed overnight while it became red.The solvent was removed under reduced pressure and the resulting residue was treated with water (100 ml) and extracted with ethyl acetate (2 x 300 ml).The organic phase was washed with brine, dried over magnesium sulfate and filtered.The solvent was removed under reduced pressure and the resulting crude was purified by silica gel column chromatography (2:1 hexane / ethyl acetate) to give a yellow oil.

ethyl N-(tert-butoxycarbonyl)-2'-[(tert-butoxycarbonyl)amino]-4-{2-[2-(tritylsulfanyl)ethoxy]ethoxy}isovalin-
ate (1.04 g, 1.4 mmol) was dissolved in a 1:1 mixture of 1 M NaOH (5 ml) and methanol (5 ml).The solution was stirred at 80 °C for 13 h.Afterwards, the solution was allowed to cool to room temperature and the pH was adjusted to pH ~ 7 with 1 M HCl.The solvent was removed under reduced pressure and the resulting residue was washed with water (4 x 10 ml) to remove the sodium chloride.Yield: 0.981 g (98%).and N,N-dimethylformamid (3 ml).The mixture was stirred at room temperature for 19 h, while a white precipitate was formed.The precipitate was separated from the solution with the help of centrifugation.

7 )
Figure S14.Radiolabelling of Au-Fe 3 O 4 Dumbbell-like NPs with an illustrative scheme of the labelling procedure (A) and the PD-10 size exclusion chromatograms after incubation at 50 °C for 60 min of NPs and 99m Tc complexes containing chelator 6 (B), 2 (C), 3 (D), 4 (E) and the corresponding control chromatograms (without reduction with TCEP).Please note that 0.5 ml fractions were collected and the very last fraction was the remainder activity in the PD-10 column (including the activity in the column).The very first 1.0 ml (two 0.5 ml fractions) is not shown in any of the chromatograms since it always was mobile phase only.

Table S1
Activity measurements of the labelling experiments of Au-Fe 3 O 4 Dumbbell-like NPs (without reduction with TCEP).