Functionalized Gold Nanoparticles as Contrast Agents for Proton and Dual Proton/Fluorine MRI

Gold nanoparticles carrying fluorinated ligands in their monolayer are, by themselves, contrast agents for 19F magnetic resonance imaging displaying high sensitivity because of the high density of fluorine nuclei achievable by grafting suitable ligands on the gold core surface. Functionalization of these nanoparticles with Gd(III) chelates allows adding a further functional activity to these systems, developing materials also acting as contrast agents for proton magnetic resonance imaging. These dual mode contrast agents may allow capitalizing on the benefits of 1H and 19F magnetic resonance imaging in a single diagnostic session. In this work, we describe a proof of principle of this approach by studying these nanoparticles in a high field preclinical scanner. The Gd(III) centers within the nanoparticles monolayer shorten considerably the 19F T1 of the ligands but, nevertheless, these systems display strong and sharp NMR signals which allow recording good quality 19F MRI phantom images at nanoparticle concentration of 20 mg/mL after proper adjustment of the imaging sequence. The Gd(III) centers also influence the T1 relaxation time of the water protons and high quality 1H MRI images could be obtained. Gold nanoparticles protected by hydrogenated ligands and decorated with Gd(III) chelates are reported for comparison as 1H MRI contrast agents.


General Information
All commercially available reagents were from Aldrich and Alfa Aesar, and used without purification unless otherwise mentioned. Solvents were purchased from Aldrich and VWR, deuterated solvents from Cambridge Isotope Laboratories and Aldrich. Dry solvents were obtained from Aldrich and Alfa Aesar. Chlorinated solvents were kept over K 2 CO 3 with occasional shaking for at least 24 h prior to use. All other solvents were reagent grade and used as received. Reactions were monitored by TLC on Merck silica gel plates (0.25 mm) and visualized by UV light, I 2 , or by KMnO 4 -H 2 SO 4 .
Nuclear Magnetic Resonance spectra were recorded on a Varian 500 spectrometer (operating at 500 MHz for proton, at 125 MHz for carbon, 470 MHz for 19 F), or on a Varian 400 MHz (operating at 400 for proton). 1 H NMR spectra were referenced to the residual protons in the deuterated solvent. 13 C NMR spectra were referenced to the solvent chemical shift. 19 F spectra were referenced to CFCl 3 .
Chemical shifts (δ) are quoted in ppm and the multiplicity of each signal is designated by the conventional abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; dd, doublet of doublets. Coupling constants (J) are quoted in Hz.

Mass spectrometry measurements were obtained by electrospray ionization (ESI) with a Perkin
Elmer APII at 5600 eV and recorded by Dr. Fabio Hollan, Department of Chemical and Pharmaceutical Sciences, University of Trieste, Italy.
UV-Visible spectroscopy measurements were recorded on a Shimadzu UV-1800 spectrophotometer.
Thermogravimetric analysis (TGA) were performed on TGA Q-500 V6.3 Build 189 using platinum pans and a heating rate of 10 °C/min up to 1000 °C or on a Netzsch STA 409 using alumina crucibles and a heating rate of 10 °C/min up to 650 °C under a static air atmosphere.
Transmission electron microscopy (TEM) images were obtained with either a Philips EM 208 operating at 100 kV or with a Joel 3010 high resolution electron microscope (1.7 nm point-to-point) operating at 300 keV using a Gatan slow-scan CCD camera (mod. 794).
Dynamic light scattering (DLS) measurements were performed on a Malvern Zetasizer Nano in backscatter mode. Analyses were performed on nanoparticle solutions with concentration of 10 mg/mL or 3.7 mg/mL, using either disposable micro cuvettes or disposable zeta cells (Malvern). The cell positioning factor was set to 0.83 mm and attenuator values of 5 or 9 were auto-optimized as a function of nanoparticles concentration.

ICP-OES:
Total Au and Gd concentrations in the samples, solubilized with 1 mL of aqua regia (mixture HCl:HNO 3 of 3:1) and diluted to 10 mL with MilliQ water, were quantified by means of Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) using an Optima 8000 instrument (PerkinElmer; USA) equipped with an S10 integrated autosampler (PerkinElmer; USA).

S3
The measurements were conducted using a calibration curve obtained by dilution of gold and gadolinium standard solutions for ICP-OES analysis (Sigma-Aldrich, Italy). The limit of detection in the obtained solutions at the operative wavelengths (Au 267.595 nm and Gd 376.839 nm) was 0.02 mg L -1 for both Au and Gd. The precision of the measurements expressed as repeatability (as RSD %) for the analysis was always less than 5%. To this mixture, a solution of NaBH 4 (0.081 g, 21 mmol) in 5.65 mL of milliQ water was added in 10 seconds and the reaction mixture was allowed to stir for 30 minutes at 0 °C and for 3 hours at room temperature. The solvent was removed and the solid residue washed with diethyl ether (5 x 30 mL), S4 the nanoparticles were transferred in two centrifuge tubes and washed with diethyl ether (5 x 30 mL).

Synthesis of
The nanoparticles purification was completed by size exclusion chromatography on Sephadex-LH20 using methanol as eluent. Solubility properties: Good solubility in water, methanol and DCM. 1

Synthesis of HS-C8-DO3A
The synthetic strategy for the preparation of the ligand HS-C8-DO3A is summarized in Scheme S2.

10-bromo-2-hydroxy-decanoic acid ethyl ester, C
To a solution of 10-bromo-2-oxo-decanoic acid ethyl ester (1.2 g, 4 mmol) in 15 mL of MeOH solid NaBH 4 was added under stirring at 0 °C. After 30 minutes, the solvent was removed then the residue was taken-up with DCM (10 mL) and the resulting solution was washed with water (1 x 5 mL). The organic phase was dried over anhydrous sodium sulfate and the solvent was evaporated affording the product as yellowish oil with 85% yield.

2-Hydroxy-10-tritylsulfanyl-decanoic acid ethyl ester, D 3
To a suspension of NaH (60% dispersion oil, 0.15 g, 3.73 mmol) in dry DMF (   NaOH 5% (2.6 mL) was added to compound TrtS-C8-DO3A t Bu, F, in 2.5 mL dioxane and the reaction was let to stir for one week adding in this period another 3 mL of NaOH 5%. Then, HCl 10 % was added and the solvent was evaporated. The solid was dissolved in 2 mL of HCl 1M and then extracted with AcOEt/MeOH 1/1 (6 mL). The organic phase was evaporated giving a yellow oil with a 15% yield. 1

Coupling of H with oxy-fluorinated tetraethylene glycol, giving the compound I
To a solution of perfluorinated tetraethylene glycol (1.383g, 3.382 mmol) in 2 mL of dry dioxane a solution of 6-tritylthio-1-paratoluenesulfonylhexane, H (0.816g, 1.537 mmol) in 2 mL of dry dioxane was added, the addition was completed in 10 minutes. To the reaction mixture, KOH pellets (0.283 g, 5.07 mmol) were added and the reaction mixture was stirred overnight at 100 o C. After the reaction was complete, the residue was dissolved in 30 mL of water and the mixture was extracted with ethyl acetate (5 x 20 mL). The organic phase was then washed with water (2 x 25 mL) and brine (2 x 25 mL) and dried over Na 2 SO 4 , filtered and evaporated under reduced pressure. The product was purified by flash chromatography using hexane/AcOEt 9/1 as eluent. Yield 56%.

TsOPEGOMe, K
A solution of TsCl (789 mg, 4.14 mmol) in 3 mL dry DCM was added drop wise to a mixture of HO-PEG-OMe (2.01 mL, 3.98 mmol) and triethylammine (1.11 mL, 7.96 mmol) diluted with 1 mL dry DCM and kept at 0 °C. The mixture was allowed to stir at room temperature for 18 hours. The

Synthesis of thiol HS-C6OF-PEG
Compound TrtS-C6OF-PEG, L (518 mg, 0.399 mmol) was dissolved in deoxygenated dichloromethane (5 mL) under argon atmosphere. To the mixture, trifluoroacetic acid (0.59 mL, 7.98 mmol) and then triisopropyl silane (0.16 mL, 0.798 mmol) were added. The reaction mixture was stirred at room temperature for 4 hours and then the solvent was removed in vacuo, the residue was co-evaporated with methanol. The crude product was purified by column chromatography over silica gel using chloroform-ethyl acetate (9:1 v/v) as eluent. Compound HS-C6OF-PEG was obtained as a colorless oil with a 97% yield.

Synthesis of HS-C6OF-DO3AGd,
This strategy for the preparation of the ligand HS-C6OF-DO3AGd is summarized in Scheme S4.

Synthesis of HS-C6OF-DO3A
Trifluoroacetic acid (0.9 mL, 11.8 mmol) was added to a solution of N (0.075 g, 0.059 mmol) in 1 mL of anhydrous DCM; the reaction mixture was allowed to stir overnight. Then the solvent was removed and to the solid residue were added 2 mL anhydrous deoxygenated DCM, 0.28 mL TFA and 0.28 mL of TIPS. The reaction was left stirring for 2h, the solvent was removed and the solid was washed five times with deoxygenated hexane. The solid was dried under argon and the product was obtained as a white solid. Yield 100 %. were added and the reaction mixture was stirred for 2 h at 25 °C. This solution was used for the place exchange reaction without further purification.  Table 1 collects T1 weighted (T1-w), T2 weighted (T2-w) and spin density weighted (SD-w) sequences key parameters.