concentration 37oC concentration 37oC of 50 µg and kept at in an incubator for 24 hours. After incubation, the particles were separated from unbound or weakly bound protein using a magnetic-activated cell sorter (MACS) and redispersed in 0.01 M salt solution for DLS and zeta potential measurements. As a control, SPIONs in water were run through the MACS column and exchanged in 0.01 M salt to check for any effects of magnetic field exposure on the size distribution. The hydrodynamic Z-average value of the control samples (Table 2) showed no significant changes compared to the original values (Table 1). When samples were exposed to medium, they formed larger assemblies that carried an overall negative charge (Table 2 & Fig S9). It should be noted that it is difficult to quote accurate Z-average values for such large structures, as data can be too poor for cumulants analysis (Figure S10). However, for all three samples, there is a clear increase in hydrodynamic radius by intensity and shift to the right of the correlation function, which is indicative of the formation of much larger structures al.21 (Figure S8). Calatayud et showed similar data for positively charged SPIONs with hydrodynamic diameter ~ 30 nm, which formed large structures with similar hydrodynamic diameters, which resulted in more uptake into neuroblastoma cells SPIONs.21 when compared to negatively charged All SPIONs here exhibited negative al.21 surface charges after exposure to medium, similar to those quoted by Calatayud et This suggests that specific proteins in the corona may induce uptake, rather than membrane.22 cationic particles binding directly to the cell SPIONs with larger cores and less polymer content in their outer shell could allow for denser packing of iron oxide into these large protein structures leading to the observed uptake values in Figure 3a. We attempted to do TGA after protein exposure and magnetic separation, but found it is very difficult to do this practically and reliably. Even if the SPIONs are eluted in deionised water only, they are still eluted with small amounts of salt, which affect the TGA when the samples are freeze dried. Methods to remove excess salts such as dialysis and spin filtering also remove some of the smaller proteins. Unfortunately therefore, we were unable to quantify the absolute amount of protein bound.

## MR Properties in Cells

We have previously suggested that the solution relaxivity of SPIONs does not tracking,10 accurately predict their efficacy for cell as the relaxivity can dramatically cells.10, 36-38 change once SPIONs are internalised into This is thought to be due to packing of SPIONs into lysosomes leading to changes in their spatial distribution as cells.10 well as the intrinsic properties of water diffusion in Most of the literature so far has focused on changes in T and T relaxation. Here, we have explored not only the 1 2 changes in T but also in T *, which is generally more sensitive to magnetic field 2 2 inhomogeneities. Because the SPIONs used here were synthesised using the same procedure, it is possible to investigate if core size has a direct effect on these properties. This is not the case when using materials from different labs and/or suppliers, where different shells and particle arrangements also need to be taken into account. Our measurements suggest that T relaxivity is strongly reduced when 2 mM-1s-1 SPIONs are internalised in cells, with values on the order of 20-23 (Figure 6a & Table S3) irrespective of the core size, compared to the solution T relaxivity 2 mM-1s-1 values in Figure 2A showed which ranged from 163-344 and increased with reports.10, 39 core size. This reduction is T is consistent with other 2 These cell relaxivity values are in very close agreement with our measurements of scanner10 commercially available materials obtained with a 7T and also with the 3T.39 values measured for cell internalised ferumoxides at Magnetic resonance images of SPIONs colloidally dispersed in agarose or internalised in MSCs are presented in