Nanosims Analysis of an Isotopically Labelled Organometallic Ruthenium(ii) Drug to Probe Its Distribution and State in Vitro

The in vitro inter- and intra-cellular distribution of an isotopically labelled ruthenium(II)-arene (RAPTA) anti-metastatic compound in human ovarian cancer cells was imaged using nano-scale secondary ion mass spectrometry (NanoSIMS). Ultra-high resolution isotopic images of (13)C, (15)N, and Ru indicate that the phosphine ligand remains coordinated to the ruthenium(II) ion whereas the arene detaches. The complex localizes mainly on the membrane or at the interface between cells which correlates with its anti-metastatic effects.

Cell preparation A2780CR cells were seeded 50000 cells/well in 24-well clear bottom plates fitted with 13mm thermanox slips.After 24 hours, cell media was aspirated and fresh media containing 15 N, 13 C, RAPTA-T 500uM was added.(Drug was diluted into media from a 20mM stock in water).Dose chosen was non-toxic 5 After 24 hours of incubation, media was aspirated, and cells were washed twice with PBS.Subsequently cells were fixed with buffered aldehydes (2% PAF, 2.5% Gluteraldehyde in PBO 1M, pH 7.4) for one hour and then washed in cacodylate buffer (0.1M, pH 7.4).After that, the cells were postfixed for 40 minutes in a solution of 1% osmium tetraoxide and 1.5% potassium ferrocyanide in cacodylate buffer.This was followed by a further staining of 1% osmium tetraoxide in cacodylate buffer, for 40 minutes, and then 1% aqueous solution of uranyl acetate for 40 minutes.The samples were then dehydrated in an ascending alcohol series (1 X 50%, 1 X 70%, 2 X 96%, 2 X 100%, 3 minutes each) and resin embedded with Durcupan resin which was then hardened overnight at 65 ⁰C.The resin embedded cells were semi-thin sectioned onto glass coverslips ready for analysis in the nanoSIMS.

Nano-SIMS analysis
NanoSIMS measurements were performed at the Laboratory of Biological Geochemistry, EPFL and the Universtiy of Lausanne.Prior to NanoSIMS imaging, the samples were gold-coated in order to avoid charging effects.Before acquiring an image, Cs ions were implanted into the surface of the sample in order to enhance the ionization of the element of interests.
In our study, the electron multiplier detectors were set up to measure 12 C 2 − , 13 C 12 C − , 12 C 14 N − , 12 C 15 N − , 31 P − , 32 S − , and 102 Ru − secondary ions, generated by bombarding the sample with a ~4 pA Cs + primary beam focused to a spot size of approximately 160 nm.
In order to resolve the possible isobaric interferences, the instrument was operated at a mass-resolving power (MRP) of about 10.000.For 102 Ru -, due to the very low signal obtained on cells, peak-shape and mass resolving power was checked using a Ru metal standard.
Data acquisition was performed by scanning the Cs + primary beam over areas of 34x34µm with a 256x256 pixel image resolution.The per pixel dwell time of the primary ion beam was 10 ms.The final images are the accumulation of 120 layers obtained by sequential scanning and correspond to a cumulated acquisition time per pixel of 1.2 seconds.Between every layer, the focusing of the secondary ion beam was optimized and automatic peak centering was performed for 12 C 2 − , 13 C 12 C − , 12 C 14 N − , 12 C 15 N − .The Ru peak could not be centered due to the low count rates.However, postanalysis check revealed that there was no significant change in the peaks position during the entire acquisition time.The total acquisition time including the centering procedure was 22 h per image.