Materials

The preparation of the complex [Os(p-cym)(1,2-dicarba-closo-dodecaborane-1,2-dilthiolate)] (1) was based on a previous report23. The triblock copolymer P123 [poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)] was purchased from Sigma-Aldrich and used as received. Anhydrous tetrahydrofuran (Aldrich) was used. Pure water (18.2 MΩ) was collected from a Purelab UHQ USF Elga system. Holey carbon grids with 200 mesh and lacey carbon grids were purchased from Quantifoil Micro Tools Gmbh and Elektron Technology UK Ltd, respectively and used as received.

Synthesis of OsMs and OsRuMs

A tetrahydrofuran solution (1 ml) of complex 1 (5 mg ml−1) was added to an aqueous solution (10 ml) of polymer P123 (5 mg ml−1) and the resultant mixture was stirred at ambient temperature for 4 h. The solution was then dialyzed to remove the tetrahydrofuran (molecular weight cutoff=1000 Da), for 48 h, and then freeze-dried. A similar procedure was used for synthesizing OsRuMs with 1 mol equivalent of 1, 1 mol equivalent of the Ru analogue and 1 mol equivalent of polymer P123.

Characterization of the micelles OsMs and P123Ms

Dynamic light scattering (DLS) experiments (Supplementary Fig. 2) unambiguously demonstrated that polymer P123 and complex 1 self-assemble in aqueous solution. Encapsulation decreased the size of P123Ms micelles from 19.6±1.80 nm (hydrodynamic diameter, D h ) to 11.5±2.35 nm for OsMs with a Ð of 0.03 (Supplementary Fig. 2a; Supplementary Table 1). Although micellar size usually increases after encapsulation of organic molecules39, incorporation of hydrophobic molecules can result in expulsion of water from the micelles, causing a contraction40. The hydrophobicity of 1 probably results in a stronger folding of the poly(ethylene oxide) chains around the complex through hydrophobic interactions, with concomitant expulsion of water from the core. A small second population of OsMs particles (<0.01% in number) was found at D h ~

220 nm, exhibiting a strong intensity in DLS analysis, owing to the aggregation of some particles (Supplementary Fig. 2c).

Cryogenic TEM (cryo-TEM) analysis without staining was then performed on Quantifoil carbon-coated grids to observe the morphology of the hydrophobic core (containing osmium complexes) of the nanoparticles in solution. The high contrast provided by the heavy osmium centres allowed facile imaging of the osmium-polypropylene glycol (PPG) core, but impaired the observation of the poly(ethylene glycol) corona owing to the polymer hydrated state; and disfavored by the small diameter of the micelles (Supplementary Fig. 3a), even after attempts to further stain the samples with uranyl acetate. From these experiments, it was clear that spherical micellar morphologies are formed when polymer P123 encapsulates complex 1. The observed diameter of these OsMs nano-spheres is 7.85±1.97 nm (Supplementary Fig. 3b) with very low close-to-ideal dispersity, based on counting 157 particles counting (1.06, 1.00 being for ideal mono-disperse systems; see Supplementary Table 1). These data are in accordance with the D h determined by DLS within experimental error.

To gain further insight into their structures in aqueous solution, and to confirm cryo-TEM and DLS results, OsMs and P123Ms were analyzed by synchrotron SAXS (Supplementary Fig. 4). The experimental profiles were fitted to three model functions for spherical micelles: SphereForm, CoreShellSphere, and PolyCoreShellRatio (PCR). The PCR model fitted excellently for both micelles with very low-Ð parameters (0.161 for OsMs and 0.146 for P123Ms, 0 being an ideal mono-disperse system, Supplementary Table 1). These analyses demonstrated that OsMs self-assembly leads to core/shell micelles with a core diameter of 9.06±0.12 nm, and a shell diameter of 6.50±0.15 nm (Supplementary Table 1). The core dimension of OsMs was larger than that of P123Ms (6.74±0.06 nm), while the corona dimension of OsMs was smaller than that of P123Ms shell (12.22±0.17 nm). The diameters of OsMs micelles by DLS and cryo-TEM are in accordance with the core diameter determined by SAXS within the experimental errors, while the diameters of P123 micelles from DLS and SAXS studies are similar.

From these data (scattering length density calculations, degrees of polymerization of Pluronic P123 and the molecular formulae of the polymer and of complex 1; see Instrumentation and Methods), aggregation numbers for OsMs and P123Ms micelles were determined as 20±2 monomer chains per P123Ms micelle and 52±6 monomer chains per OsMs micelles. Determinations of osmium by ICP-MS gave a polymer/complex 1 ratio of 1/1±0.091 for OsMs showing that the 52 chains polymer chains self-assembled with 52±11 complexes 1 (see Supplementary Table 1). Similar core/shell diameters (Supplementary Table 1) with excellent fits to the PCR model were obtained from experiments at three different concentrations (1, 5, 10 mg ml−1; Supplementary Fig. 4a,b) for both OsMs and P123Ms. Hence concentration does not influence the micellar structure, a parameter of importance for Pluronic-type self-assemblies.

Instrumentation

Ultraviolet-visible spectroscopy

Ultraviolet-visible absorption spectra were recorded on a temperature-controlled Varian CARY 300 Biospectrophotometer using 1-cm path-length quartz cuvettes (0.5 ml).

Inductively coupled plasma mass spectrometry

Osmium (189Os) content was determined using an ICP-MS Agilent technologies 7500 series instrument. The standard for osmium was purchased from Aldrich. Calibration curves were prepared using Os standard solutions in double deionised water with 3% nitric acid, ranging between 50 and 0.5 ppb (9 points). Samples were freshly prepared in double deionised water with 3% nitric acid. Readings were made in no-gas mode with a detection limit of 1 ppt for 189Os.

Dynamic light scattering

The D h of nanoparticles was determined by DLS. Typically, an aqueous nanoparticle solution was measured with a Malvern Zetasizer NanoS instrument equipped with a 4 mW He-Ne 633 nm laser module at 25 °C. Measurements were carried out at a detector angle of 173° (back scattering). Data were analyzed by the Malvern DTS 6.20 software. D h was calculated by fitting the apparent diffusion coefficient in the Stokes–Einstein equation D h =kT/(3πηD app ), where k is the Boltzmann constant, T is the temperature and η is the viscosity of the solvent. D h coincides with the hydrodynamic diameter when the sample is made of monodispersed spherical particles (D app equals the translational diffusion D t ).

Transmission electron microscopy

TEM was performed using a JEOL 2000FX operating at 200 kV. TEM samples were prepared by using holey and lacey carbon grids. One drop of the sample solution (5 μl) was applied to the grid and after 2 min the solution was blotted away before drying. Images were recorded on a Gatan Orius camera and were analyzed using ImageJ software. At least 100 particles from different parts of the grid were counted for each sample to obtain the average diameter.

Cryogenic-transmission electron microscopy

A JEOL 2010F TEM was operated at 200 keV and images were recorded on a Gatan UltraScan 4000 camera for cryo-TEM and glow discharge. The samples were prepared at ambient temperature by placing a droplet on a TEM grid. The extra liquid was then blotted with a filter paper and the grid was inserted in liquid ethane at its freezing point. The frozen samples were subsequently kept under liquid nitrogen.

High-resolution electron microscopy

A JEOL JEM-ARM200F HR-TEM was operated at 80 keV, 1.9 pA cm−2, with spherical aberration (C s ) tuned to approximately +1 μm and images were recorded on a Gatan SC1000 Orius CCD camera.

Small-angle X-ray scattering

Measurements were carried out on the SAXS beamline at the Australian Synchrotron facility at a photon energy of 11 keV. The samples in solution were in 1.5 mm diameter quartz capillaries. The measurements were collected at a sample to detector distance of 3.252 m to give a q range of 0.004–0.2 Å−1, where q is the scattering vector and is related to the scattering angle (2θ) and the photon wavelength (λ) by the following equation (1):