Gadolinium chloride hexahydrate ([GdCl3, 6H2O], 99.999%), sodium hydroxide (NaOH, 99.99%), tetraethyl orthosilicate (Si(OC2H5)4, TEOS, 98%), aminopropyl-triethoxysilane (H2N(CH2)3-Si(OC2H5)3, APTES, 99%), triethylamine (TEA, 99.5%), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 99.5%), sodium hydroxide (NaOH, 99.99%), sodium chloride (NaCl, 99.8%), calcium chloride (CaCl2, 99%), the bovine serum albumin and dimethyl sulfoxide (DMSO, 99.5%) were purchased from Aldrich Chemicals (France). Diethylene glycol (DEG, 99%) was purchased from SDS Carlo Erba (France). Acetone (reagent grade) was purchased from Sodipro (France) and was used in the same conditions as received. 1,4,7,10-tetraazacyclododecane-1-glutaric anhydride-4,7,10-triacetic acid (DOTAGA) was furnished by CheMatech (Dijon, France). Gadolinium oxide cores were furnished by Nano-H S.A.S (Saint-Quentin Fallavier, France). Only milli-Q water was used for the preparation of aqueous solutions of nanoparticles.
DLS (Dynamic Light Scattering) size measurement
Direct measurements of the size distribution (in DEG) or after dilution (at 10 mM in [Gd3+] for water) of the nanoparticles were performed via Zetasizer NanoS DLS (laser He-Ne 633 nm) from Malvern Instrument.
Direct determination of the ζ-potential of the hybrid nanoparticles were performed via a Zetasizer NanoS from Malvern Instruments. Prior to the experiment, the nanoparticles were diluted in an aqueous solution containing 0.005 M in NaCl and adjusted to the desired pH.
Inductively coupled plasma-atomic emission spectrometry (ICP-AES) analysis
Determination of the gadolinium content in a sample was performed by ICP-AES analysis (with a Varian 710-ES spectrometer). Before measuring gadolinium concentration, samples of colloidal solution were dissolved in concentrated nitric acid for 24 hours. The samples were then diluted with water, until the nitric acid concentration in water reached 5%. Chemical analyses were also performed on the as-prepared samples at the Service Central d’Analyses du CNRS (Solaize, France) by ICP-AES, and enabled determining the C, N, Si contents with a precision of 0.5%.
Relaxation time measurements were performed using a Bruker Minispec MQ60 NMR analyser, operating at 1.4 T magnetic field.
High performance liquid chromatography (HPLC)
Gradient HPLC analysis was done by using Shimadzu Prominence series UFLC system with a CBM-20A controller bus module, a LC-20 AD liquid chromatograph, a CTO-20A column oven and a SPD-20A UV-visible detector. UV-visible absorption was measured at 295 nm. 20 μL of sample were loaded in the solvent injection ratio: 95% solvent A – 5% solvent B (A = Milli-Q water/TFA 99.9:0.1 v/v; B = CH3CN/Milli Q water/TFA 90:9.9:0.1 v/v/v) onto a Jupiter C4 column (150 × 4.60 mm, 5 μm, 300 Å, Phenomenex) at a flow rate of 1 mL/min over 5 min. In a second step, samples were eluted by a gradient developed from 5 to 90% of solvent B in solvent A over 15 min. The concentration of solvent B was maintained over 5 min. Then, the concentration of solvent B was decreased to 5% over a period of 5 min to re-equilibrate the system, followed by additional 5 min at this final concentration. Before each sample measurement, a baseline was performed following the same conditions by loading Milli-Q water into the injection loop.
Preparation of gadolinium oxide cores []
A solution was prepared by dissolving 167.3 g of [GdCl3, 6 H2O] in 3 L of DEG at room temperature. The solution is then stirred for 3 hours at 140°C. Afterwards, 44.5 mL of sodium hydroxide solution (10 M) is added to the solution. This final solution is stirred for 5 hours at 180°C before cooling and stirring for 12 hours at ambient temperature. The gadolinium oxide cores displayed hydrodynamic diameters of 1.7 ± 0.5 nm.
Encapsulation of gadolinium oxide cores by polysiloxane
The polysiloxane shell is ensured by sol–gel process by addition of silane precursors. First, a solution containing 1.6 L of DEG, 51.42 mL of TEOS and 80.61 mL of APTES is slowly added (during 96 hours) to the precedent solution under stirring at 40°C. One hour after the end of the addition of the silane precursors, a second solution of 190 mL of DEG, 43.1 mL of water and 6.94 mL of TEA is added under stirring at 40°C during 96 hours. At the end of the second addition, the solution is stirred for 72 hours at 40°C and finally for 12 hours at ambient temperature. The gadolinium oxide cores coated by the polysiloxane displayed hydrodynamic diameter of 2.6 ± 1.0 nm.
Covalent grafting of DOTAGA on the nanoparticles
163.7 g of DOTAGA anhydride are then added to the core-shell nanoparticles in DEG. The resulting solution is stirred for 72 hours at room temperature.
17.5 L of acetone are added to the solution to precipitate the nanoparticles that are filtered under vacuum before dispersion in water. The solution is stirred for one hour at pH 2 before remaining acetone is removed by evaporation. The nanoparticles are then purified by tangential filtration through 5 kDa membrane. The solution is stirred at pH 5 for 12 hours before another tangential filtration through 5 kDa membrane after addition of sodium hydroxide solution to reach pH 7.4. Afterward, the solution is filtered twice through a 1.2 μm and then 0.2 μm syringe filters to remove the largest impurities. Finally, the nanoparticles are freeze-dried and can be stored for months without alterations. After dispersion in water, the AGuIX nanoparticles display hydrodynamic diameter of 2.2 ± 1 nm. Elementary analysis found (weight percent) C, 26.84; N, 7.79; Gd, 13.83; Si, 10.79. An average mass of 8.7 ± 1 kDa is obtained by mass spectrometry. The longitudinal relaxivity (r1) is 11.5 mM−1.s−1 at 1.4 T. The nanoparticles display an isoelectric point at pH 7.5 ± 0.5. About 50 g of nanoparticles are obtained at the end of the synthesis. HPLC chromatogram, with UV absorption detection at 295 nm, shows a retention time of 10 min for the nanoparticles.
Imaging and MRT procedure
All operative procedures related to experiments on glioma bearing rats strictly conformed to the French government regulations with licenses 380825 and B3818510002 and were approved by the internal evaluation committee for animal welfare and rights of the ESRF. All operative procedures related to MR on healthy mice strictly conformed to the Guidelines of the French Government with licenses 380324 and A3818510002 for MRI on healthy mice. All experiments were performed under anesthesia with the following parameters: 5% isoflurane for induction and intraperitoneal injection of xylazine/ketamine (64.5/5.4 mg.kg−1) for maintenance.
Brain tumor inoculation
The 9 L gliosarcoma (9LGS) cells were implanted in the brain of male fisher F344 rats (Charles River, France) []. Anesthetized animals were placed on stereotactic frame, and 104 9LGS cells were suspended in 1 μL culture medium with antibiotics before to be injected through a burr hole in the right caudate nucleus (3.5 mm lateral to the bregma, 6 mm below the skull surface).
Preparation of injectable solution
After tangential filtration, a concentrated colloid (AGuIX in water, [Gd3+] = 100 mM) was diluted by aqueous solution containing NaCl and hepes in order to obtain an intravenous use solution ([Gd3+] = 40 mM, [NaCl] = 145 mM, [hepes] = 10 mM). The pH was adjusted to 7.4. Before use, this solution was filtered onto syringe filter with nylon membrane (pore diameter 0.22 μm). The chelate used was DOTAREM® (laboratories Guerbet, Aulnay sous Bois France, 0,5 mM/mL) as available in MRI units.
The aqueous AGuIX ([Gd3+] = 40 mM, [NaCl] = 145 mM, [hepes] = 10 mM) colloid was manually injected in the saphena vein at 1.4 mL volume using a 2 mL syringe and a 26 G needle. The gadolinium chelates were injected via the saphena vein according 2 protocols. First, a series of rats received an injection of a colloidal solution at the concentration of 1 M in Gd , i.e. twice the concentration routinely used in clinical conditions (0.5 mM). The volume injected was of 56 μL which corresponds to a quantity of Gd (56 μmol) similar to those currently injected. Second, another series of rats received a 1.4 mL DOTAREM® injection diluted in physiological serum until 40 mM (to establish a direct comparison with the nanoparticles, the quantity of Gd injected being the same than previously).
The MR imaging took place at Grenoble Institute of Neuroscience (GIN) using a 7 Tesla Imaging system (Biospec, Bruker, Erlangen Germany) equipped with a 400 mM/T gradient. The images were made 14 days after implantation, and the rats were injected in the saphena vein with 1.4 ml of a Gd based particles solution at 40 mM in gadolinium. Images were acquired at fixed times after injection using a T2 weighted Turbo RARE SE sequence (TR = 4,000 ms, TE = 33 ms, FOV = 3 cm, Resolution = 0,12 mm, ST = 1 mm) and a T1 weighted FLASH sequence (TR = 840 ms, TE = 10,804 ms, FOV = 3 cm, Resolution + 0,12 mm, ST = 1 mm).
Radiation source and microbeam radiation therapy (MRT) set-up
Irradiations were performed at the ID17 Biomedical Beamline of the European Synchrotron Radiation Facility (ESRF, France) using X-rays emitted tangentially from electron bunches circulating in a storage ring. The wiggler produces a wide spectrum of photons which extends, after filtration, from 50 over 350 keV (median energy: 90 keV). The mean dose rate was then 62 Gy.mA−1.s−1 allowing very fast irradiation. The quasilaminar beam was micro-fractionated into an array of 41 rectangular and quasi-parallel 50 microns width microbeams, separated by 200 microns centre to centre. The setup was performed by using the ESRF Multislit Collimator, positioned 33 m from the photon source, and 80 cm upstream from the rat holder. Ten days after tumor inoculation, the animals were positioned prone on a Kappa-type goniometer (Huber, Germany) in front of the X-rays source, on a homemade Plexiglas frame, and the alignment into the beam was performed using live cameras. The contention of the rats was performed by a teeth bar, while the animals were under anaesthesia. They were received a lateral irradiation, from their anatomical right to left side, followed by an antero-posterior irradiation (cross fired configuration). The beam was shaped into a field of irradiation of 10 mm horizontal, and the animals were scanned vertically over 10 mm through the beam after opening of the shutter. Although the total procedure lasted about 2 min, for each rat, the irradiation time is around 2 s. Animal immobility during exposure was checked on three control video screens located in the control hutch. The microbeam dose at the tumor (i.e. 7 mm of depth from lateral side) was 400Gy, the valley dose was 18.6 Gy as computed by Monte Carlo simulations. The spatial configuration of irradiation was checked by radiochromic films (Gafchromic, HD-810) exposed in front of rats.
The survivals of animals were represented on Kaplan − Meier curves and compared using the log-rank test; the Median Survival time (MeST) postimplantation was calculated (Prism, GraphPad Software, San-Diego, USA).