Nanoscale hydrophilic colloids with high relaxivity and low cytotoxicity based on Gd ( III ) complexes with Keplerate polyanions

1. Materials and methods 1.1. Materials Commercial chemicals Gd(NO3)3·6H2O (99.9wt%), NaOH (pellets, 98.5wt%), anhydrous (C2H5)2O (99.7 wt%), N2H4·H2SO4 (99 wt%), (NH4)6Mo7O24·4H2O, Pluronic F-127 (suitable for cell culture), Pluronic F-68 (suitable for cell culture), Pluronic P-123 were purchased from Sigma Aldrich. Absolute C2H5OH (99.5%), CH3COONH4 (98 wt%), ammonium hydroxide (2830 wt%), H2SO4 (96 wt%) were obtained from Acros Organics. The purchased chemicals were used as received without further purification. Cell culture WI-38 VA 13 subline 2RA (human embryo lung) was obtained from collection of the institute of cytology of Russian Academy of Sciences. The standard nutrient medium “Igla” with an addition of 10% calf serum and 1% of indispensable amino acids was purchased from Institute of poliomyelitis and viral encephalitis named after M.P. Chumakov (Moscow, Russia).

used as received without further purification.
Cell culture WI-38 VA 13 subline 2RA (human embryo lung) was obtained from collection of the institute of cytology of Russian Academy of Sciences.The standard nutrient medium "Igla" with an addition of 10% calf serum and 1% of indispensable amino acids was purchased from Institute of poliomyelitis and viral encephalitis named after M.P. Chumakov (Moscow, Russia).

Synthesis of the Gd x (Kp) y -based colloids.
The colloids were synthesized by simple mixing of the aqueous solutions of the triblock copolymers 9.92 mL for F-127 (0.081 mM), F-68 (0.121 mM) and P-123 (0.501 mM) with 0.07 mL of (NH 4 ) 42 Kp (1 mM) with the following admixture of 0.015 mL of Gd(NO 3 ) 3 (55.6 mM) under the stirring.

Methods
Raman spectra were recorded on a  Small angle X-Ray scattering (SAXS) data for samples were collected with the Bruker AXS Nanostar SAXS system using CuK α (λ 1.5418Å) radiation from a 2.2 kW X-ray tube ( 35kV, 40 mA) coupled with Gobbel mirrors optics and a HiStar 2D area detector.The beam was collimated using three pinholes with apertures of 800, 450 and 700 μm.The instrument was operated with a sample-to-detector distance of 63.9 cm to provide data at angles 0.1º< 2θ < 4.8º, which correspond to 0.007 Å −1 < q < 0.34 Å −1 .The value of q is proportional to the inverse of the length scale (q = (4π/λ)sin(θ) in units of Å −1 ).Scattering patterns were obtained for the samples at 23˚C in an evacuated chamber.The measurements were performed in transition mode with the use of glass capillaries filled by liquid samples.The capillaries (2 mm diameter) were sealed and TEM images were obtained by use of Hitachi HT7700 (Japan) at an accelerating voltage of 100 kV.

Cell viability evaluation
Cell viability of human embryo lung cells (WI-38 VA 13 subline 2RA) towards studied nanoparticles was determined by means of multifunctional system Cytell Cell Imaging (GE Healthcare Life Sciences, Sweden) using application Cell Viability BioApp.The cells were dispersed in culture medium at the cell conc.200000 cells per ml.Then, the cells were seeded into 96-well plates at 150 lor 30 × 10 3 cells per well and cultivated in CO 2 -incubator at 37°C for 24 hours.After removing the culture medium and their redispersion in the fresh culture medium the cells were exposed by 50 l of F-127-Gd x (Kp) y , P-123-Gd x (Kp) y , F-68-Gd x (Kp) y colloids in accordance with Cell Viability Bio App protocol, were Gd(III) and Kp concentrations were adjusted at 0.08, 0.04 and 0.02 mM for Gd(III), 0.018, 0.0088 and 0.0044 mM of Kp.Thus, 30 × 10 3 cells per well were exposed by 0.02, 0.01 and 0.005 mM of Gd(III), 0.0045, 0.0022 and 0.0011 mM of Kp.The concentrations of the triblock copolymers in the cellular samples were varied within 0.02, 0.01 and 0.005 mM for F-127, 0.125, 0.0625, 0.0313 for P-123, 0.03, 0.015, 0.0075 mM for F-68.After 45 minute exposure the percentage of viable Wi-38 cells was calculated with the use of Cytell Cell Imaging system.The experiments were repeated three times.The intact cells cultivated simultaneously with the studied ones served as a reference.The fraction of the grown-up cells was expressed in % vs. reference cells.

Relaxometric measurements
The transverse relaxation times T 2 of water molecule protons in studied solutions were measured The relative measurement deviation for transverse and longitudinal relaxation times does not exceed 3%.Longitudinal and transverse relaxivities r 1,2 are defined as: where (1/T i ) meas is the value measured for the sample with concentration C (mM) of magnetic centres, (1/T i ) dia refers to the nuclear relaxation rate of pure water protons, r 1 , r 2 are longitudinal and transverse relaxivities, respectively.
T 1 and T 2 relaxation times of water protons were also measured by means of whole body 1.5 T scanner (Excel Art Vantage Atlas X, Toshiba, Japan) equipped with 65-cm horizontal bore size corresponding to a proton resonance frequency of 63.58 MHz.The special NMR sample tubes (Wilmad glass company, INC) were inserted in the centre of 20 cm cubic phantom filled with distilled water.
All T 1 and T 2 measurements were recorded at 23-25C.
The dilution of the synthesized colloids was applied for the variation of Gd(III) concentration from 0.08 to 0.02 mM for plotting1/T 1 and 1/T 2 versus Gd(III) concentration in the Fig. 3.      Figure S8.Guinier plot (log I versus q 2 ) of the SAXS data measured for the samples 1-4.
Guinier plots (log I versus q 2 ) of the SAXS data (Fig. S8) indicates the presence of linear regions in all curves.The radius of gyration Rg was deduced from a weighted linear regression of the scattering curves.Under the assumption of the spherical symmetry of the particles, the average radii R NP (R NP = (5/3)R g ) of the particles are 127.7,131.6, 144.5 and 119.3Å for samples 1 to 4, respectively (the samples designation is from Fig. S8).It is obvious that the increase in particle size and their interaction with each other is accompanied by the shifting to smaller angles of the peaks in the scattering curves (Fig. S7), which indicates the increased distances between the particles centers.The impact of the slowed down rotation regime in the high relaxivity of F-127-Gd x (Kp) y is confirmed by low relaxivity (5 mM -1 •s -1 ) for F-127-Tb x (Kp) y (Fig. S9a, b, ESI), where electron spin reorientation ( s ) is the correlation time.
Room-temperature data were collected in the reflection mode with a flat-plate sample.The sample was applied in liquid form on the surface of standard zero diffraction silicon plate.After drying the layer applied on top of it a few more layers were placed to increase the total amount of the sample.The sample was kept spinning (15 rpm) throughout the data collection.Patterns were recorded in the 2θ range between 3 o and 100 o , in 0.008 o steps, with a step time of 0.1-4.0s.Dynamic light scattering (DLS) measurements were performed by means of the Malvern Mastersize 2000 particle analyzer.A He-Ne laser operating at 633 nm wavelength and emitting vertically polarized light was used as a light source.The measured autocorrelation functions were analyzed by Malvern DTS software and the second-order cumulant expansion methods.The effective hydrodynamic radius (RH) was calculated by the Einstein-Stokes relation from the first cumulant: D = kBT/6πηRH, where D is the diffusion coefficient, kB is the Boltzmann constant, T is the absolute temperature, and η is the viscosity.The diffusion coefficient was measured at least three times for each sample.The average error in these experiments is approximately 4%.The samples for DLS study were prepared from deionized water, sonicated for 60 minutes and equilibrated at 25.0±0.1°Cbefore DLS and zeta-potential measurements.
put into evacuated chamber by means of the holders.For each sample, two experiments were performed, allowing to control the quality of the experiments.The results of two experiments are summarized, so that the total time of each experiment was equal to 50000 s.Integration of twodimensional pictures scattering was performed using a software package SAXS [Small Angle Xray Scattering.Version 4.0.Software Reference Manual, 2000, M86-E00005-0600, Bruker AXS Inc.].Data analysis performed by programs SASView [S1] and PRIMUS [S2].The UV-vis measurements were conducted on Lambda 35 spectrophotometer (Perkin-Elmer, USA) using a 10 mm cuvettes at room temperature.pHs of the solutions were controlled with Microprocessor pH meter «pH 212» (Hanna Instruments, Germany) The pH-meter was calibrated with standard aqueous buffer solutions (pHs 7.01 and 4.01).
using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence[S3].The longitudinal relaxation times T 1 of water molecule protons were defined with the use of the analyzer's inversionrecovery pulse sequence with 20 data collected for fitting[S4].The experiments were performed at 25±0.5 °C on an MQ20 Minispec NMR Analyzer (Bruker, Germany) at a field strength of 0.47 T (19.65 MHz).The classical inversion-recovery method using 180°-τ-90° pulse sequence was performed to determineT 1 .The repetition delay was chosen to be at least five times T 1 .The dead time was 7 μs.Four scans were acquired for each measurement.T 2 was measured with a Carr-Purcell-Meiboom-Gill (CPMG) sequence with a 90-180° pulse gap of 1.0 ms.The 90° pulse was 2.64 μs and 180° pulse was 5.16 μs in length.

Figure
Figure S1.(left) The structure of Kp, estimated by X-ray.(right) Raman spectra of Kp -in solution (black) and in solid state (red).

Figure S2 .
Figure S2.Raman spectrum of Gd 14 Kp in solid state.

Figure S4 .
Figure S4.The experimental X-ray powder diffraction patterns of powder sample of ammonium salt of Kp (blue curve) and dried sample of the Gd x (Kp) y colloids (red curve).
Electronic Supplementary Material (ESI) for New Journal of Chemistry.This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2017 C 60 H 517 O 704 Mo 132 Gd 14 : calcd: C 2.6,