MATERIALS AND METHODS:
Chemicals:
Gadolinium chloride hexahydrate ([GdCl
3
.6H
2
O], 99%, Nano-H France), sodium hydroxide
(NaOH, 99.99%, Sigma Aldrich), diethylene glycol (DEG, >99%, SDS), tetraethylorthosilicate (Si(OC
2
H
5
)
4
, TEOS, 98%, Sigma Aldrich), (3aminopropyl)triethoxysilane (H
2
N(CH
2
)
3
-Si(OC
2
H
5
)
3
, APTES, 99%, Sigma Aldrich),
1,4,7,10-tetraazacyclododecane-1-glutaric anhydride-4,7,10-triacetic acid (DOTAGA,
CheMatech ®
), triethylamine (TEA, 99%, Sigma Aldrich), anhydrous dimethylsulfoxyde
(DMSO, Sigma Aldrich), Pre-activated Cyanine 5.5(Cy5.5-NHS, GE Healthcare
®
).
Size measurements and surface potential:
Hydrodynamic diameters (HD) and ξ -potentials of our samples were determined with a
ZetasizerNanoS PCS (Photon Correlation Spectroscopy, Laser He-Ne 633 nm) from Malvern
Instruments ®
.
Fluorescence measurements:
Fluorescence measurements were carried out at room temperature using a Varian Carry
Eclipse fluorescence spectrophotometer.
High Performance Liquid Chromatography:
Gradient HPLC analysis was carried out using the Shimadzu ®
Prominence series UFLC system equipped with a CBM-20a controller bus module, an LC-20AD liquid chromatograph, a CTO-20A column oven and SPD-20AUV-visible detector. Sample UV-visible absorption was measured at 295 nm.Sample aliquots of 20 μL were loaded in 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 x 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 30 min. The concentration of solvent B was maintained over 10 min. Then, the concentration of solvent B was decreased to 5 % over a period of 10 min to re-equilibrate the system, followed by additional 10 min at this final concentration. Before each sample measurement, a baseline was performed following the same conditions byloading Milli-Q water into the injection loop.
Nanoparticles synthesis:
The synthesis of the gadolinium based nanoparticles is a four step synthesis. The first step is the synthesis of a gadolinium oxide core by addition of soda on gadolinium chloride salts in
DEG [32]. The second step is the growth of a polysiloxane shell by addition of adapted

amounts of TEOS and APTES in DEG. Then DOTAGA anhydride is covalently grafted on the free amino functions of APTES and during the transfer from DEG to water of the particles a top down process is observed with a dissolutionof the gadolinium oxide core due to the chelation by DOTAGA. This lead to the ultrasmall gadolinium based nanoparticles made of a polysiloxane matrix surrounded by gadolinium chelates [18, 19].
Preparation of gadolinium oxide cores:
Gadolinium chloride salt (167.3g, GdCl
3
.6H
2
O) was placed in 3 L of diethylene glycol (DEG) at room temperature under vigorous stirring. The suspension was heated at 140°C until the total dissolution of gadolinium chloride salt (about 1 h). When the solution is clear, sodium hydroxide solution (120 mL, 3.38 M) was added drop by drop under vigorous stirring.
Afterwards, the solution was heated and stirred at 180°C for 3h. A transparent colloid of gadolinium oxide (Gd
2
O
3
) nanoparticles (HD = 1.6±0.5 nm) was obtained([Gd] = 30 mM)and can be stored at room temperature for weeks without alteration.
Coating of gadolinium oxide cores by a polysiloxane shell:
The previously obtained Gd
2
O
3 colloidal solution is diluted in DEG to obtain a final volume of
4.5 L ([Gd] = 30 mM). The silane precursors (APTES (75.8 mL)) and TEOS (48 mL)) and hydrolysis solution (aqueous Et
3
N in DEG (0.75M of TEA, 75M of water) are continuously added to the precedent solution over a four day period under stirring at 40°C (HD= 3.5±0.7 nm). After the end of the addition, the final mixture was stirred for 48H at 40°C (HD =
5.7±1.0 nm).
Functionalization of the polysiloxane shell by DOTAGA:
To perform the functionalization of the core-shell nanoparticles (core: Gd
2
O
3
/ shell: polysiloxane)DOTAGA anhydride (108.9 g diluted in 500 mL of anhydrous DEG) is added to
3.25 L of the precedent solution during a period of three days under strirring.
Purification and dissolution of the gadolinium oxidecore:
The precedent solution is precipitated in 14L of acetone before filtration and washing by 2x2L of acetone. A white powder is obtained that is dispersed in water and purified by tangential filtration to a 1000 factor over a 5 kDa membrane (HD = 4.1±1.0 nm at pH 7.4). The nanoparticles are then freeze-dried for storage, using a Christ Alpha 1-2 lyophilizer. The freeze dried nanoparticles are stable for months without alterations.
Grafting of Cy 5.5 near infrared dye:
To perform optical imaging, Cy 5.5 near infrared dye was grafted on the nanoparticles.Cy5.5-
NHS was diluted in anhydrous DMSO (2.5 mg/L) and added to an aqueous solution of freshly dispersed USPRs in water at pH 7-7.4 for 8 hours under stirring (molar ratio Cy5.5/Gd
0.044/1). Nanoparticles were then submitted to tangential filtration to a 1000 factor over a 5 kDa membrane before freeze-drying.

RESULTS
The USRPs are synthesized by a previously described protocol [18, 19] slightly modified to obtain higher quantities of nanoparticles. It consists first in the synthesis of gadolinium oxide core by addition of soda on gadolinium trichloride previously dissolved in DEG. Then the growth of a polysiloxane shell is ensured by the addition of adapted amount of silane precursors (TEOS and APTES). Nanoparticles presenting a HD of 5.7 ±1.0 nm are obtained at this step. DOTAGA anhydride is then covalently grafted on the free amino functions issued from APTES. During the transfer to water of these nanoparticles, an original top down process occurs: the gadolinium issued from the core is chelated by the DOTAGA and then a collapse and a fragmentation of the polysiloxane shell into ultrasmall nanoparticles, made of polysiloxane and surrounded by about ten DOTA chelates, is observed as previously described. When needed for optical imaging experiments, Cy 5.5 was grafted on the USRPs as described in the experimental section.
The nanoparticles present a HD of about 4.1 ± 1.0 nm. The particles have rather a neutral charge at physiological pH, namely a value of zeta potential of -0.2 mV at pH = 7.4. The number of free DOTAGA on the nanoparticles has been determined by adding increasing amounts of europium chloride and by evaluating the fluorescence of the chelated europium ions [19]. Less than 5% of free DOTAGA are present on the nanoparticles maximizing the relaxivity of each nanoparticle. The r
1
per nanoparticle is equal to 8.22 mM -1 s -1 per gadolinium ion at 4.7T. Finally, the purity of the nanoparticles has been evaluated more than
95% by HPLC.