## Results and Discussion SPION synthesis and properties

The labelling of stem cells with SPIONs of different polymer coatings and surface charges is relatively well established, with cationic SPIONs generally being oxide.11, 13 the most efficient in increasing the overall internalisation of iron However, the influence that the mass fraction of polymer in a SPION shell can have on the uptake by stem cells is still not well understood or indeed reported. SPIONs with different polymer contents can be synthesised by simply changing the amount of al.,23 polymer in the co-precipitation reaction. Basuki et showed that using less polymer in a co-precipitation reaction resulted in SPIONs with larger core sizes and lower percentage polymer content in the SPION shell. Here, we have used a similar approach but employed amine-containing FITC-DEAE-dextran polymer to yield cationic SPIONs with very similar surface charge. We then investigated the efficiency of these SPIONS for stem cell labelling and imaging using MR. SPIONs were synthesised with different core sizes and polymer content (Scheme 1) using the co-precipitation approach as described previously by our group,16 al.25 which is a modification of methods used previously by Molday et and al.26 Paul et The three samples in this study were synthesised by using polymer to iron salt ratios of approximately 4:1, 1:1 and 1:4, whilst all other experimental parameters were kept constant. A 2:1 mass ratio of FeCl 6(H O)/ FeCl 4(H O) was employed as 3. 2 2. 2 it has been previously shown to lead to iron oxide cores with a high (> 95%) magnetite content.27 After each synthesis, the SPIONs were dialysed (using 100 kDa membrane) to neutralise the pH and then concentrated using 100 kDa spin filters before passing them through Sephadex® G-100 beads. mL-1 To obtain a concentrated, sterile stock solution of 1 mg [Fe basis] for cell labelling, SPIONs were concentrated again and passed through a sterile 0.22 µm polyethersulfone membrane. This thorough cleaning procedure was applied to remove any polymer not bound to the SPION surface. Decreasing the amount of FITC-DEAE dextran polymer in the co-precipitation reaction resulted in SPIONs that had very similar surface charges (all between 16.9 – 19.4 mV, Table 1), but lower polymer content and larger core sizes (Table 1, Figure 1) as measured by TGA and pXRD respectively (Figures S1 & S2). Our measurements show hydrodynamic diameters of 40 to 60 nm in 0.01M NaCl and 83-86 nm in distilled water (Table 1, Figures S3 & S4), which is the medium in which SPIONs were stored. Transmission electron microscopy images (TEM) show clear differences in particle core size (Fig S5), although for core size calculations pXRD was preferred to TEM, as it can provide distribution.28 more statistically accurate particle size However, we highlight that the method used to determine the core sizes from the pXRD data is such that the values should not be taken as absolute. There was an expected correlation between SPION core size, solution relaxivity 2T,29 s-1 at 9.4T and magnetisation at with T relaxivity increasing from 163 mM for 2 s-1 the SPIONs with the smallest core size up to 245 and 344 mM for SPIONs with a core size of 8.4 and 9.9 nm respectively (Figure 2a). The T * relaxivity followed the 2 same trend and was slightly higher than that obtained for T with values of 171, 285 2 s-1 and 454 mM for SPIONs of 6.7, 8.4 and 9.9 nm respectively (Figure 2b). Magnetisation at 2T also increases with particle size (Figure 2c), with 6.7 nm particles g-1, g-1 displaying a saturation magnetisation of 101 emu 8.4 nm particles 108 emu and g-1 9.9 nm 120 emu by extrapolating high field magnetisation data to zero (Table S2 & g-1 Figure S6). All values are quoted is emu [Fe] as the exact phase purity of samples is unknown. mL-1 [Fe],11, 16 Using a nominal labelling concentration of 50 µg we measured the colloidal stability in water and cell culture medium by UV-vis over a period of 1