Supplementary information

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1 Supplementary information Optimization of PEG coating conditions Coating conditions were optimized in order to address the full effect of different lengths of PEG polymer. A ligand exchange was made in toluene with a silane amine (3-aminopropyltrimethoxysilane, Si-NH 2 ) together with PEG molecules with NHS ester end groups (α-methoxy-ω- NHS esters, NHS-PEG). The amine group on the Si-NH 2 reacts with the NHS ester of the NHS-PEG and form an amide bond to covalently link the Silane and PEG molecules. The amount of Si-NH2 and the ratio of Si-NH 2 to NHS-PEG (2000 Da) were varied and added to 20 mg oleic acid coated iron oxide nanoparticles. The Si-NH 2 amount was varied from µl to 6 µl and the molar ratio between NHS-PEG and Si-NH 2 (NHS-PEG/Si-NH 2 ) was varied between 0.75 and 3. The stability was determined by measuring the iron content that remained in suspension after centrifugation of purified particles (Figure S1a). For particles with the lowest ratio of NHS-PEG to Si- NH2 (NHS-PEG/Si-NH2 = 0.75), significant aggregation (stability < 50%) was observed for both low and high amounts of Si-NH 2. Aggregation of these particles was expected because the amount of NHS- PEG molecules was lower than the amount of Si-NH2 molecules resulting in a combination of Si-PEG and frees Si-NH 2 on the particle surface. The two particles formulations coated with a higher NHS- PEG/Si-NH 2 ratio (1.5 and 3) aggregated in the presence of low amounts of Si-NH 2, and the stability increased at higher amounts of Si-NH 2. The most stable particle exhibited a stability of 92% and was achieved at the NHS-PEG/Si-NH 2 ratio of 1.5 and with 6 µl Si-NH 2 added. As a control devoid of PEG, nanoparticles were coated with Si-NH 2 ( µl) in the absence of NHS-PEG and expectedly these particles showed very poor stability (below 1%). To test for nonspecific non-covalent PEG coating, other nanoparticles were made with NHS-PEG (6.3 and 100 mg). But in the absence of Si-NH 2, both particles formulations aggregated with stability below 5%. Nanoparticles coated with commercial Silane-PEG molecules were used as a positive control, and these particles formulation showed a high stability of 83%.

2 Figure S1 Stability of PEG coated nanoparticles after centrifugation. The ratio of iron before and after centrifugation is shown as %. (a) Stability of MNPs as a function of Silane amine (Si-NH2) and at different ratios of PEG to Silane (NHS-PEG/Si-NH2) (b) Stability of particles coated with variable amounts of Silane amine (Si-NH2), PEG (NHS-PEG) alone or Silane-PEG (Si-PEG).

3 The RAW macrophage cell line was used as a model for phagocytic capture of the synthesized MNPs and predicts interaction with the MPS system. This uptake should be low in order to get the best accumulation of particles in the tumors [26, 49]. Cells were incubated with 0.1 mg [Fe]/ml for 24 hours and washed thoroughly before measuring the total amount of iron and protein in the cells using ferrozin and Bradford assay, respectively. The uptake of nanoparticles in the cells was measured as the ratio between iron and protein. The uptake was higher for nanoparticles synthesized with a low amount of Si-NH2 (0.375µl) compared to higher amounts (3 and 6 µl Si-NH 2 ) (Figure S2). The ratio of NHS- PEG/Si-NH2 was important, as nanoparticles with 1.5 µl silane amine and a low ratio (1.5) had a higher uptake than nanoparticles coated with higher ratios. All particles coated with Si-NH 2 amounts higher than 3 µl and a NHS-PEG/Si-NH2 ratio of 1.5 or 3 showed low uptake. Control particles coated with PEG in the absence of silane amine showed a high uptake; between 2-3 times higher than the most stable particles formed in the presence of silane amine. Nanoparticles without PEG showed a very high uptake whereas, nanoparticles coated with the commercial silane-peg showed very low uptake (Figure 3). We conclude that uptake in macrophages cells correlate with the tendency of the particles to aggregate. The preference for the macrophages to take up larger particles is consistent with previous reports [35, 50, 51]. The phagocytotic uptake by macrophages can give an indication of the unspecific uptake by the reticuloendothelial system and should preferably be kept low in order to get the best accumulation of particles in the tumors [26, 49].

4 Figure S2 Uptake in RAW cell line. (a) Uptake of particles with different amounts of Silane amine and at two different PEG to Silane ratios. (b) Uptake of particles coated only with Silane amine (Si-NH2), only PEG (NHS-PEG) or Silane-PEG (Si-PEG). Based on these result, we decided to synthesis NMPs using 6 µl Si-NH 2 together with a NHS-PEG/Si- NH 2 ratio of 1.5, as these particles showed good stability and low uptake in macrophage.

6 In the high resolution C 1s spectrum the component with binding energy around ev correspond to C-C-O (PEG) and C-C-N (Si-NH 2 ). Since PEG is the major component and significant proportion of amine groups of Si-NH 2 react to form amide linkage (288 ev), it is valid to neglect the contribution from C-C-N to the component at ev. The C 1s high resolution spectra revealed that the contribution from alkane groups were significantly higher for the particles coated with the low Mw PEG compared to particles coated with high Mw PEG, with the PEG of Da as an exception. The alkane peak could come from oleic acid remaining on the particles after coating with silane. However Si-NH 2 and carbon contaminations also influence the alkane contribution, which could explain the large alkane contribution for the particles coated with Da PEG. Calculating inelastic mean free path of Fe 2p photoelectron The inelastic mean free path (IMFP) value of Fe 2p photoelectron through PEG was estimated using the scheme developed by Cumpson et al. [39] based on a quantitative structure property relationship (QSPR). The QSPR enables the estimation of IMFP of 1 kev electron through any polymer with a known chemical structure. To begin with, the molecular index of polymer repeat unit at the zero-order, o v, is calculated using the valence connectivity index values [39]. Once o v is known, the IMFP value of photoelectrons with kinetic energy E kev can be calculated by using equation S1. Cumpson et al. had reported the value of IMFP for 1 kev photoelectrons through PEG using the above mentioned procedure [39]. Cumpson et al. also showed that by using equation S2, it is possible to calculate the IMFP value for electrons of kinetic energy E kev in a polymer, if the IMFP for 1 kev electrons is known [39]. Here we calculated the IMFP of Fe 2p photoelectrons with kinetic energy 780 ev through PEG to be 2.46 nm using equation S2 based on the IMFP value of 1 kev photoelectrons reported by Cumpson et al. [39]. [ ] Eqn-S1 Eqn-S2

7 Where, λ i attenuation length of electrons of kinetic energy E kev, take off angle of analyzer, o v atomic connectivity index of polymer repeat unit, N rings number of aromatic rings, N non-h number of atoms in repeat unit excluding hydrogen atoms, E KeV kinetic energy of photoelectron. λ i attenuation length of electrons of kinetic energy 1 kev.

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