Diffusion of nanomedicines inside the extracellular matrix (ECM) has been identified as a key factor to achieve homogeneous distribution and therefore therapeutic efficacy. Here, we sought to determine the impact of nanoparticles surface properties on their ability to diffuse in the ECM. As model nano-objects, we used a library of gold nanoparticles grafted with a versatile polymethacrylate corona which enabled to modify the surface properties. To accurately recreate the features of native ECM, diffusion studies were carried out in a tumor-derived gel (Matrigel®). We developed two methods to evaluate the diffusion ability of NPs inside this model gel: an easy to implement one based on optical monitoring and another one using small-angle X-ray scattering (SAXS) measurements. Both enabled to determine the diffusion coefficients of NPs and compare the influence of their various surface properties, while the SAXS technique also allowed to monitor the NPs structure as they diffused inside the gel. Positive charges and hydrophobicity were found to particularly hinder diffusion, and the different results suggested on the whole the presence of NPs-matrix interactions, therefore underlying the importance of the ECM model. The accuracy of the tumor-derived gels used in this study was evidenced by in vivo experiments involving intratumoral injections of NPs on mice, which showed that diffusion patterns in the peripheral tumor tissues were quite similar to the ones obtained within the chosen ECM model.