week. The absorbance was measured at 400 nm and normalised to t = 0. When SPIONs sediment, this is not only visually noticeable, but also reflected by a drop in absorbance. There was no loss in absorbance over a period of seven days for SPIONs suspended in water at room temperature or in cell culture medium (DMEM + 10 % FCS) at 37 °C (Figure S7 A&B), showing that there was no sedimentation under storage or labelling conditions. Photographs of the cuvettes at day 7 are shown in Figure S7C, demonstrating the absence of sediment.

## Cellular Uptake of SPIONs and impact on Viability and Function

We evaluated the uptake and viability of each of the SPIONs into murine 3).10, 16 mesenchymal stem cells using a labelling period of 24 hours (Figure Intracellular iron levels were dependent on the exposure concentration, but even though samples had very similar hydrodynamic properties, their uptake profiles mL-1 increased significantly as the core size increased (Figure 3a). At 50 µg [Fe], the mass of iron uptake was 4.4 pg [Fe]/cell for the sample with 6.7 nm mean core size, increasing to 12.9 pg[Fe]/cell for the sample with 8.4 nm mean core size and 21.5 pg[Fe]/cell for the sample with 9.9 nm mean core size, representing a near 5-fold increase in uptake. The cell viability measured using an ATP assay (Figure 3b) is independent of the mass of iron oxide taken up by MSCs. There is a slight drop in mL-1 viability at higher labelling concentrations (50 and 100 µg [Fe]) for all samples but, interestingly, the observed decay in ATP levels was dependent on the exposure dose rather than on the amount of iron oxide internalised by cells (Figure 3b). Increasing the uptake of SPIONs into stem cells without altering viability is a common challenge.11 synthetic We have shown this is possible by simply decreasing the amount of polymer in the reaction. The data shown in Figure S7 supports that these uptakes are related to physicochemical properties of SPIONs, not due to their sedimentation. Importantly, cells were able to proliferate and differentiate in vitro after mL-1 being labelled with SPIONs at a concentration of 50 µg [Fe]. Alizarin red staining, which is specific for calcium deposits, and Oil red staining, which reveals the presence of lipid droplets, were present in all samples after an induction period of 7 days, irrespective of the SPION labelling (Figure 4). Prussian blue stained images of SPION-labelled MSCs (Figure 5) show a noticeable difference in intracellular iron mL-1 content at a labelling concentration of 50 µg [Fe], with the staining intensity correlated with the amount of intracellular iron measured at this concentration (Figure 3a). To assess whether the increased uptake was reflected in the MR contrast of 105 mL-1 labelled MSCs, 3 x cells were labelled at a concentration of 50 µg [Fe], suspended in agarose and imaged using a T weighted sequence. For all cells labelled 2 with SPIONs, a strong negative contrast is observed in relation to unlabelled cells. The loss of signal is proportional to the uptake data presented for each sample (Figure 5 & insets). The loss in intensity as quantified using imaging software is shown in Figure S8. Recently, more quantitative studies have explored how charge and polymer formed,30-34 type can affect the nature of the protein corona that is as well the role SPIONs.35 surface charge and protein corona can have on the biological fate of One of the most challenging obstacles for investigating how the protein corona can change hydrodynamic charges and surface charge of nanomaterials is the extraction of medium.32 nanoparticles after exposure to It is difficult to successfully extract nanoparticles plus protein corona, without washing away some of the weakly bound proteins or aggregating particles irreversibly. However, information on changes in size and surface charge during labelling could help understand differences in the extent of cell uptake for different nanomaterials. To understand why core size could lead to such remarkable differences in uptake, we investigated how the properties of SPIONs change after exposure to medium. SPIONs were suspended in culture medium at a