The in vivo characterization of Gadolinium (Gd) based MRI Contrast Agents (MR-CA) diffusion within brain extracellular space after a transient and local Blood Brain Barrier permeabilization induced by ultrasound is of great interest for the understanding of drug transport in brain parenchyma in the framework of new pharmaceutical developments for Central Nervous System diseases.Four Gd-chelates with different hydrodynamic diameters (d_H) were tested: three commercially available MR-CA (MultiHance, Gadovist and Dotarem) and a new class of Gd-based nanoparticles (AGuIX, University of Lyon). These latest compounds are composed of a core of polysiloxane, grafted with two or three Gadolinium chelates. They are sufficiently small (d_H <5nm) to escape hepatic clearance and can be easily functionalized¹. Diffusion Light Scattering (DLS) measurements were performed to estimate the hydrodynamic diameter of all compounds (Table 1). The MRI acquisitions were performed with a 7T/90 mm Pharmascan scanner (Bruker). To evaluate the CAs’ longitudinal relaxivities (r₁) at 7T and 37°C, bundles of tubes containing different CA-concentrations in 0.3 % w/w agar gel were prepared. The T₁ values of these tubes were measured using an IR-FGE sequence²(T_E/T_R1= 2.5/5 ms, 6 segments, 90 TI from 75ms to 8975ms, FA =5°, resolution = 0.250 x 0.250 x 1.25 mm³, delay between two segments T_R2 = 15000ms, NA = 6). Measured values of 1/T₁ for different CA-concentrations [CA] were fitted using the following equation³: 1/T₁=1/T₁₀+r₁x[CA], where T₁₀ is the T₁ of the media without Gd (see Fig.1). Relaxivity values are summarized in Table 1 for all compounds. The Free Diffusion Coefficients (D_Free) of these compounds were estimated by injecting 10µL of a 5mM solution in a tube filled with 0.3 % w/w agar gel. The diffusion was followed for 1 hour by acquiring five T₁-maps (IR-FGE, T_E/T_R1 = 2.5/5 ms, 6 segments, 60 TI from 88ms to 5100ms, FA = 5°, resolution = 0.225x0.225x1 mm³, T_R2 = 9000ms, NA = 1, total duration = 12.5min). The tubes were kept at 37°C during the acquisition. A T₁₀-map acquired before the injection was used as a reference. MR-CA concentration maps were then calculated from T₁-maps using the previous equation. On each CA map, a 2D Gaussian function was fitted⁴. The square of the widths of the Gaussian fit was plotted along time and fitted with the relationship σ²_x,y= 2t x D_x,y,vitro for both σ_x and σ_y. For each compound D_Free was estimated as the average D_Free= (D_x,vitro+ D_y,vitro)/2. Hydrodynamic diameter (d_H) of the CA was deduced from D_Free using the Stokes Einstein formula D_Free=kT/(3πηd_H) where k = 1.38x1023 Pa.m³.K^-1 is the Boltzmann constant, T is the temperature in Kelvin degrees and η is the viscosity of the agar gel(6.92·10^-4 Pa.s). Focal ultrasound induced BBB permeabilization was also performed⁵ in the right striatum of 11 Sprague-Dawley rats (120g, Janvier, France), followed by intravenous MR-CA injections. The Gd chelates diffusion starting from the BBB disruption site was followed by repeatedly acquiring T₁-maps for about 1 hour, as for the in vitro measurements. The same Gaussian fitting procedure was applied and the Apparent Diffusion Coefficients (ADC) of all the compounds in the striatum were estimated as the average ADC = (D_x,vivo+ D_y,vivo)/2.Figure 2a shows examples of in vitro CA-maps obtained for MultiHance. The fitted 2D-Gaussian functions are presented in Fig.2b whereas in Fig.2c the linear fit on their spreads is plotted. Fig.3 shows an example of in vivo dataset acquired by injecting Dotarem in one rat. In Fig.3a the original CA maps acquired within 66 minutes after the CA injection are pictured, and their respective Gaussian fits are shown in Fig.3b. The linear fit over these Gaussian spreads is given in Fig.3c. As can be noticed in Table 1, both the D_Free and the ADC are decreasing with increasing hydrodynamic diameters ( Dotarem > Gadovist > MultiHance > AGuIX). Furthermore, quantitative values of hydrodynamic diameters deduced from D_Free measurements are really consistent with DLS measurements (see Table 1).The ADC values have been used to estimate tissue tortuosity $$$\sqrt{\frac{D_{Free}}{ADC}}$$$ (Table 1), showing a very good agreement with the tortuosities evaluated with more standard techniques⁶. This agreement confirms the validity of this method to estimate the ADC values in the tortuous regime, but also that the diffusion properties of brain tissue are not altered by the chosen blood-brain barrier permeabilization protocol unlike by direct intracerebral injection⁴.